Chronic Hepatorenal Toxicity Induced by Low-Dose Co-Exposure to Chlorpyrifos and Bisphenol A in Wistar Rats: Protective Effects of Hesperidin

Chronic Hepatorenal Toxicity Induced by Low-Dose Co-Exposure to Chlorpyrifos and Bisphenol A in Wistar Rats: Protective Effects of Hesperidin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chronic Hepatorenal Toxicity Induced by Low-Dose Co-Exposure to Chlorpyrifos and Bisphenol A in Wistar Rats: Protective Effects of Hesperidin Enokela Shaibu Idoga, Nendirmwa Musa Dashe, Onuche Shalom Agweche, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9163020/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : The extensive use of pesticides and the increasing production and consumption of bisphenol A found in plastics pose new challenges regarding toxicity and environmental pollution. The study aimed to evaluate the protective effects of hesperidin on hepato-renal and oxidative stress changes provoked by chronic co-exposure of rats to bisphenol A (BPA) and chlorpyrifos (CPF). Thirty male Wistar rats were assigned into six groups of five rats each: Group I (C/oil) were administered Corn oil (2 mlkg -1 ), II (CPF) and III (BPA) were administered chlorpyrifos (4.75mgkg -1 ; 1/20 th LD 50 ) and bisphenol A (LOAEL; 50 mgkg -1 day -1 ), respectively, while group IV (CPF+BPA) was co-exposed to CPF (4.75 mgkg -1 ) and BPA (50mgkg -1 day -1 ). Group V (HES) was administered hesperidin (100 mgkg -1 ), while group VI (CPF+BPA+HES) rats were pretreated with HES (100mgkg -1 ) and then co-exposed to CPF (4.75mgkg -1 ) and BPA (50mgkg -1 day -1 ) 30 minutes later. The different treatments were administered orally once daily for 16 weeks. At the end of the experiment, blood samples were collected and the serum was used to assess various health markers. These included total protein, albumin, urea, creatinine, and C-reactive protein levels, as well as the activity of certain enzymes like aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). Additionally, the levels of antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured, along with the levels of malondialdehyde (MDA). The liver and kidney tissues were also examined for any signs of damage. Results : The results showed that exposure to both BPA and CPF caused significant changes in these biochemical parameters and damaged the liver and kidneys. However, supplementation with HES helped to reduce the harmful effects of co-exposure to BPA and CPF, suggesting a potential protective role. Conclusions : Low dose of the combination of the CPF and BPA resulted in marked alterations in the parameters evaluated, and hesperidin offered some level of protections to the liver and kidneys of exposed rats. This protective effects may be due to its anti-inflammatory and the antioxidant ability to scavenge reactive oxygen species generated by the contaminants. Bisphenol A Chlorpyrifos Oxidative stress Hesperidin Antioxidant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Environmental contaminants from industrialization pose significant challenges to public health and the environment as they are capable of causing harm to humans, animals, and ecosystems (Grandjean and Landrigan, 2014). These contaminants encompass a variety of substances including pesticides, heavy metals, radioactive elements, and endocrine disruptors like bisphenol A (Manzetti et al., 2014). Bisphenol A (BPA) is a hazardous chemical that is extensively produced, widely utilized as a synthetic product worldwide and is commonly found in food packaging, medical devices, feeding bottles, and various plastic items (Katiyar et al., 2024; Omodon et al., 2024; Pelletier et al., 2026). The preponderate use of BPA has the capacity to cause damage to humans, animals and the environment. Exposure to BPA occurs chiefly via ingestion, inhalation, and transdermal routes (Nayak et al., 2022). Among these, the consumption of BPA-contaminated food, water, and beverages represents the most significant pathway of entry into the body (Helal et al., 2013; Begum et al., 2020; Adiga et al., 2022). Given BPA’s high global production volume and its tendency to leach from plastic products, exposure among both humans and animals has become pervasive (Steffenson et al., 2020). Among all endocrine disruptors, BPA is one of the most manufactured synthetic substances globally (Costa et al., 2025). Bisphenol A has emerged as a compound of significant public health concern, because people can be exposed to it in many ways, and even small amounts can cause harm (Konieczna et al., 2015). Organophosphates (OPs) represents one of the most extensively used classes of pesticides, accounting for approximately 50% of all pesticides used globally (Wołejko et al., 2022). The persistent presence of OP residues in the environment poses significant public health concerns worldwide as these residues can be found in food and water. Developing nations, such as Nigeria, bear a disproportionately higher burden of pesticide poisoning due to unregulated exposure from consumption of contaminated food and water (Bala et al., 2017). Chlorpyrifos (CPF) is an OP insecticide with wide application in agriculture and public health. Its primary mode of causing harm is by irreversibly inhibiting acetylcholinesterase (AChE), an enzyme essential for the smooth running of the nervous system (Sakinah et al., 2024; Coppola et al., 2025). In addition to affecting AChE, CPF has also been linked to inducing oxidative stress and endocrine disruption, further expanding its spectrum of molecular toxicity. These secondary effects can further contribute to the toxicity and multisystem harm caused by exposure to this insecticide (Rathod and Garg, 2017). Oxidative stress is a major cause of many health problems, including diseases that affect the brain and nervous system, heart and blood vessels, and other parts of the body. It's also linked to diabetes, cancer, and problems that come with getting older (Liguori et al., 2018). Moreover, exposure to environmental pollutants and xenobiotics such as pesticides like CPF (Uchendu et al., 2015) and BPA (Amjad et al., 2020) has been shown to induce oxidative stress. Hesperidin (HES) is a potent bioflavonoid and naturally occurring antioxidant. Flavonoids are small molecular-weight compounds with phenolic structures found in medicinal plants and food items. They are abundant in nature and serve as one of the predominant bioactive constituents in citrus fruits (Noshy et al., 2022). Beyond its antioxidant capacity, HES also has a range of other benefits, including reducing inflammation, lowering blood pressure, fighting cancer, and protecting against cell damage (Pyrzynska, 2022; Osama et al., 2024), thus positioning it as a promising multifunctional bioactive compound with broad therapeutic potential. The extensive use of pesticides and the increasing consumption of BPA found in plastics pose new challenges regarding toxicity (hepato- and nephrotoxicity) and environmental pollution. Real-world environmental exposures to both humans and animals rarely occur in isolation; rather, they typically involve simultaneous or sequential exposure to multiple chemical agents, raising critical concerns about cumulative and potentially synergistic toxic effects of such co-exposures. Despite this reality, the overwhelming majority of toxicological research and safety assessments have historically focused on single chemicals in isolation. While such studies yield valuable data for individual chemical risk assessment, they fall critically short in capturing the complex toxicodynamic interactions and cumulative health burdens arising from co-exposure to chemicals in humans and animals (Bloch et al., 2023). People are exposed to a wide range of chemicals every day through food, drinks, cosmetics, and indoor and outdoor pollutants. Studying the combined effects of low doses of multiple chemical contaminants on humans and animals is essential due to their widespread presence in the environment. It is also important to identify substances that may help reduce the negative health effects on the liver and kidneys following chronic co-exposure to low doses of these pollutants. As far as we know, this is the first time these two contaminants are being studied in combination. Materials and methods Experimental animals Thirty male Wistar rats, eight weeks old and weighing between 150 and 180 g, were sourced and housed in the Animal Holding Facility of the Veterinary Teaching Hospital, University of Jos, Jos, Nigeria. Prior to the commencement of the experiment, the animals were allowed a minimum acclimatization period of two weeks under standard laboratory conditions. Throughout the study, the rats were maintained on standard rat chow with unlimited access to clean drinking water. The experiment was done by following the rules set by the Institutional Animal Care and Use Committee of Animal Experimental Unit, Department of Pharmacology, University of Jos (UJ/FPS/F17-00379), as well as the guidelines for Care for Laboratory Animals (NRC 1996). 1996). Chemical Source Chlorpyrifos, which is 20% emulsifiable concentration, sold as Termiphos® (Sabero organic, India) and corn oil (Wesson Corn oil®, U.S.A) were procured from reputable stores in Nigeria, while bisphenol A and hesperidin purchased from Sigma Aldrich, U.S.A. Experimental design Thirty rats were randomly divided into six equal groups of five animals each. Group I (C/oil) was administered corn oil at 2 mlkg -1 , while group II (CPF) was administered chlorpyrifos (CPF) at 4.75mgkg -1 (corresponding to 1/20 th LD 50 previously established by Uchendu et al. (2014). Group III (BPA) was administered Bisphenol A at 50 mgkg -1 day -1 (LOAEL for BPA in mammalian studies [Vandenberg et al., 2013]), while group IV (BPA +CPF) was co-administered BPA at 50mgkg -1 day -1 and CPF at 4.75mgkg -1 . Group V (HES) was administered Hesperidin only at 100 mgkg -1 , while group VI (HES+CPF+BPA) was pretreated with hesperidin at 100mgkg -1 and then co- exposed to CPF at 4.75mgkg -1 and BPA at 50mgkg -1 day -1 . All treatments were administered via oral gavage once daily for a duration of 16 weeks. At the end of the 16-week treatment period, the animals were humanely euthanized following light anaesthesia using ketamine at 50mgkg -1 , and the blood sample was collected from each rat by jugular venesection. A small amount of blood, about 3 mL of blood was taken from each animal into a test tube. The blood was then left to clot at room temperature. After that, it was spun around at 800 x g in a centrifuge for 10 minutes. The serum samples was carefully taken out and put into new, clean test tubes. These tubes were then stored at -4 o C until they were ready to be analyzed for any biochemical changes. Serum biochemical assay The serum samples were used to determine the concentrations of total protein, albumin, urea, and creatinine; aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities using a serum biochemistry autoanalyzer, Selectra XL (Netherlands). Globulin was derived by subtracting the values of albumin from total protein. C-reactive protein concentration determination C-reactive protein (CRP) is a liver-derived acute-phase reactant that is released into circulation after tissue injury, infection or inflammation. The concentration of CRP was assayed in the serum samples using rat CRP ELISA kit (Abcam, UK) according to the manufacturer’s instruction. Determination of oxidative stress biomarkers The activities of serum antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)) were assessed using commercially available kits from Northwest Life Science Specialties Vancouver, LLC, Canada. Serum SOD activity was measured using the method described by Martin et al. (1987). To assess SOD activity, we monitored the autoxidation rate of haematoxylin. By comparing the autoxidation rates in the presence and absence of the serum, we were able to determine SOD activity. The absorbance was measured at 560 nm every 10 minutes. The results were expressed as McCord-Fridovich 'cytochrome c' units. Serum CAT activity was determined according to the method of Beers and Sizer (1952) and expressed as IUL -1 . The activity of GPx in the serum was evaluated based on the method adapted from Paglia and Valentine (1967). In this method, GPx catalyzes the oxidation of glutathione by cumene hydroperoxide. The reduction in absorbance of nicotinamide adenine dinucleotide phosphate (NADPH) was measured at 340 nm in the presence of glutathione reductase and NADPH. To measure the amount of lipid peroxidation, MDA in the serum, the double heating method developed by Draper and Hadley (1990) and later modified by Yavuz et al. (2004) was used. This technique relies on spectrophotometric quantification of the chromogen formed by the reaction between thiobarbituric acid (TBA) and MDA. Absorbance was recorded at 532 nm with an ultraviolet spectrophotometer. The MDA concentration was calculated using the absorbance coefficient for the MDA-TBA complex, 1.56×10 5 CM -1 M -1 and reported as nanomoles per milliliter. Histopathological Analysis Kidney and liver samples were harvested at necropsy and fixed in 10% buffered formalin for 48 h. The tissues were subsequently processed and embedded in paraffin blocks using tissue processing procedures, as described by Luna (1968). Very thin slices about 4 μm thick were taken from each block and stained with hematoxylin and eosin (H&E). The stained sections were then looked at under a light microscope (Leica DM 1000, Germany) under x400 magnification. Data analysis All data are presented as mean ± standard error of the mean (SEM). To see if there are any differences between groups, one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test applied for multiple comparisons were used. All the data were analyzed using GraphPad Prism, version 5.0 for Windows (GraphPad Software, San Diego, California, USA). A probability value of P < 0.05 was considered significant. Sometimes, we also looked at percentages to show how things changed, when it was helpful to do so. Results Experimental animals Thirty male Wistar rats, eight weeks old and weighing between 150 and 180 g, were sourced and housed in the Animal Holding Facility of the Veterinary Teaching Hospital, University of Jos, Jos, Nigeria. Prior to the commencement of the experiment, the animals were allowed a minimum acclimatization period of two weeks under standard laboratory conditions. Throughout the study, the rats were maintained on standard rat chow with unlimited access to clean drinking water. The experiment was done by following the rules set by the Institutional Animal Care and Use Committee of Animal Experimental Unit, Department of Pharmacology, University of Jos (UJ/FPS/F17-00379), as well as the guidelines for Care for Laboratory Animals (NRC 1996). 1996). Chemical Source Chlorpyrifos, which is 20% emulsifiable concentration, sold as Termiphos® (Sabero organic, India) and corn oil (Wesson Corn oil®, U.S.A) were procured from reputable stores in Nigeria, while bisphenol A and hesperidin purchased from Sigma Aldrich, U.S.A. Experimental design Thirty rats were randomly divided into six equal groups of five animals each. Group I (C/oil) was administered corn oil at 2 mlkg -1 , while group II (CPF) was administered chlorpyrifos (CPF) at 4.75mgkg -1 (corresponding to 1/20 th LD 50 previously established by Uchendu et al. (2014). Group III (BPA) was administered Bisphenol A at 50 mgkg -1 day -1 (LOAEL for BPA in mammalian studies [Vandenberg et al., 2013]), while group IV (BPA +CPF) was co-administered BPA at 50mgkg -1 day -1 and CPF at 4.75mgkg -1 . Group V (HES) was administered Hesperidin only at 100 mgkg -1 , while group VI (HES+CPF+BPA) was pretreated with hesperidin at 100mgkg -1 and then co- exposed to CPF at 4.75mgkg -1 and BPA at 50mgkg -1 day -1 . All treatments were administered via oral gavage once daily for a duration of 16 weeks. At the end of the 16-week treatment period, the animals were humanely euthanized following light anaesthesia using ketamine at 50mgkg -1 , and the blood sample was collected from each rat by jugular venesection. A small amount of blood, about 3 mL of blood was taken from each animal into a test tube. The blood was then left to clot at room temperature. After that, it was spun around at 800 x g in a centrifuge for 10 minutes. The serum samples was carefully taken out and put into new, clean test tubes. These tubes were then stored at -4 o C until they were ready to be analyzed for any biochemical changes. Serum biochemical assay The serum samples were used to determine the concentrations of total protein, albumin, urea, and creatinine; aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities using a serum biochemistry autoanalyzer, Selectra XL (Netherlands). Globulin was derived by subtracting the values of albumin from total protein. C-reactive protein concentration determination C-reactive protein (CRP) is a liver-derived acute-phase reactant that is released into circulation after tissue injury, infection or inflammation. The concentration of CRP was assayed in the serum samples using rat CRP ELISA kit (Abcam, UK) according to the manufacturer’s instruction. Determination of oxidative stress biomarkers The activities of serum antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)) were assessed using commercially available kits from Northwest Life Science Specialties Vancouver, LLC, Canada. Serum SOD activity was measured using the method described by Martin et al. (1987). To assess SOD activity, we monitored the autoxidation rate of haematoxylin. By comparing the autoxidation rates in the presence and absence of the serum, we were able to determine SOD activity. The absorbance was measured at 560 nm every 10 minutes. The results were expressed as McCord-Fridovich 'cytochrome c' units. Serum CAT activity was determined according to the method of Beers and Sizer (1952) and expressed as IUL -1 . The activity of GPx in the serum was evaluated based on the method adapted from Paglia and Valentine (1967). In this method, GPx catalyzes the oxidation of glutathione by cumene hydroperoxide. The reduction in absorbance of nicotinamide adenine dinucleotide phosphate (NADPH) was measured at 340 nm in the presence of glutathione reductase and NADPH. To measure the amount of lipid peroxidation, MDA in the serum, the double heating method developed by Draper and Hadley (1990) and later modified by Yavuz et al. (2004) was used. This technique relies on spectrophotometric quantification of the chromogen formed by the reaction between thiobarbituric acid (TBA) and MDA. Absorbance was recorded at 532 nm with an ultraviolet spectrophotometer. The MDA concentration was calculated using the absorbance coefficient for the MDA-TBA complex, 1.56×10 5 CM -1 M -1 and reported as nanomoles per milliliter. Histopathological Analysis Kidney and liver samples were harvested at necropsy and fixed in 10% buffered formalin for 48 h. The tissues were subsequently processed and embedded in paraffin blocks using tissue processing procedures, as described by Luna (1968). Very thin slices about 4 μm thick were taken from each block and stained with hematoxylin and eosin (H&E). The stained sections were then looked at under a light microscope (Leica DM 1000, Germany) under x400 magnification. Data analysis All data are presented as mean ± standard error of the mean (SEM). To see if there are any differences between groups, one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test applied for multiple comparisons were used. All the data were analyzed using GraphPad Prism, version 5.0 for Windows (GraphPad Software, San Diego, California, USA). A probability value of P < 0.05 was considered significant. Sometimes, we also looked at percentages to show how things changed, when it was helpful to do so. Results Effect of treatments on liver function enzymes The AST activity in the CPF group was significantly higher (P < 0.05) than in the C/oil group. Significant (P < 0.05) elevation was also observed in the BPA and CPF+BPA groups when compared to C/oil group. The AST activity in the CPF + BPA group was significantly higher (P < 0.05) than in the HES group. Notably, there was a significant decrease (P < 0.05) in the CPF + BPA+HES group compared to the CPF + BPA group. Similarly, the ALT activity in the BPA group was significantly higher than in the C/oil group. The activity in the CPF + BPA group was also significantly higher (P 0.05) in the activity of ALT between CPF+BPA and CPF+BPA+HES groups, however, there was a 19.6% decrease in the CPF + BPA + HES group compared to the CPF + BPA group. There were no significant differences in ALP activity between the treatment groups. Although there was no significant difference (P > 0.05) between CPF+BPA and CPF+BPA+HES groups, it decreased by 15.7% in the CPF + BPA + HES group compared to the CPF + BPA group (Fig 1). Effect of treatments on urea concentration The concentration of urea was significantly higher (P < 0.05) in the CPF + BPA group compared to those of C/oil, HES, CPF and BPA groups, respectively. Notably, there was a significant decrease (P < 0.05) in the CPF + BPA+ HES group compared to the CPF + BPA group (Fig. 2). Effect of treatments on creatinine concentration Creatinine concentration was significantly increased (P < 0.05) in the CPF + BPA group compared to the C/oil, HES, BPA and the CPF + BPA + HES groups, respectively. There was a significant decrease (P < 0.05) in the CPF + BPA + HES group compared to the CPF + BPA (Fig. 3). Effect of treatments on serum proteins Serum total protein concentration was significantly elevated (P < 0.05) in the C/oil group when compared to the CPF group. Similarly, a significant increase (P 0.05) in serum total protein concentration between CPF+BPA and CPF+BPA+HES groups. The serum albumin concentration was significantly increased (P 0.05) between CPF+BPA and CPF+BPA+HES groups, however, there was a 10. 5% increase in the albumin concentration in the CPF + BPA + HES group compared to the CPF + BPA group. There was a significant increase (P < 0.05) in the concentration of serum globulin in the CPF +BPA group compared to the C/oil, BPA, HES and CPF + BPA + HES groups, respectively. Notably, there was a significant decrease (P < 0.05) in the group treated with CPF + BPA + HES compared to the group treated with CPF + BPA (Fig. 4). Effect of treatments on C - reactive protein concentration The serum C-reactive protein concentration was significantly lesser (P < 0.05) in the C/oil group compared to the CPF, BPA and CPF + BPA groups, respectively. Similarly, there was significant increase (P < 0.05) in the CPF compared to the HES and CPF + BPA + HES groups, respectively. The concentration of C- reactive protein also significantly increased (P < 0.05) in the BPA, CPF + BPA and CPF + BPA + HES groups compared to the HES group. Notably, a significant decrease (P <0.05) was observed in the CPF + BPA + HES group compared to the CPF + BPA group (Fig. 5). Effect of treatments on malondialdehyde concentration The amount of MDA was much higher (P < 0.05) in the CPF group compared to the other groups, like the ones that got C/oil, BPA, CPF + BPA, HES, and CPF + BPA + HES. The amount of MDA was significantly lower (P < 0.05) in the groups that got HES and CPF + BPA + HES compared to the group that got CPF + BPA (Table 1). Effect of treatments on the activities of antioxidant enzymes Catalase activity exhibited a significant decrease in the CPF, BPA, CPF + BPA and CPF + BPA + HES groups (P < 0.05) compared to the C/oil group. Similarly, the activity also decreased significantly (P < 0.05) in the CPF group compared to the BPA, HES and CPF + BPA + HES groups, respectively. It was also noted that activity in the BPA group was much lower (P < 0.05) than in the CPF + BPA and HES groups. A significant (P < 0.05) decrease was recorded in the CPF + BPA and CPF + BPA + HES groups compared to the HES group. Notably, the activity in the CPF + BPA + HES group increased significantly (P < 0.05) compared to the CPF + BPA group (Table 1). The GPx activity in the CPF group significantly decreased (P < 0.05) compared to that of the C/oil, HES, and the CPF + BPA + HES groups, respectively. Similarly, the GPx activity in the HES group was significantly higher (P 0.05) between CPF+BPA and CPF+BPA+HES groups, it increased by 61.8% in the CPF+BPA+HES group compared to the CPF+BPA group (Table 1). The SOD activity was significantly decreased in the CPF and CPF + BPA groups (P < 0.05) compared to the C/oil group. In addition, the activity was much lower (P < 0.05) in the CPF group than in the HES and CPF + BPA + HES groups. The SOD activity also rose significantly (P 0.05) difference in the activity between CPF+BPA and CPF+BPA groups, it was elevated by 46.8% in the CPF+BPA+HES group compared to the CPF+BPA group (Table 1). The GPx activity in the CPF group significantly decreased (P < 0.05) compared to that of the C/oil, HES, and the CPF + BPA + HES groups, respectively. Similarly, the GPx activity in the HES group was significantly higher (P 0.05) between CPF+BPA and CPF+BPA+HES groups, it increased by 61.8% in the CPF+BPA+HES group compared to the CPF+BPA group (Table 1). The SOD activity was significantly decreased in the CPF and CPF + BPA groups (P < 0.05) compared to the C/oil group. Furthermore, the activity was significantly lower (P < 0.05) in the CPF group compared to the HES and CPF + BPA + HES groups, respectively. The SOD activity also increased significantly (P 0.05) difference in the activity between CPF+BPA and CPF+BPA groups, it was elevated by 46.8% in the CPF+BPA+HES group compared to the CPF+BPA group (Table 1). Histopathological findings The liver tissue of the corn oil (C/oil) and hesperidin (HES) groups showed normal histological appearance with hepatocellular architecture intact (Fig. 6. 1,5). Examination of liver tissue in rats given CPF showed mild loss of hepatocytes (L) and mild focal infiltration of inflammatory cells (IC) (Fig. 6. 2). Moderate congestion (C), mild loss of hepatocytes (L) and moderate accumulation of eosinophilic material in the hepatic vein (E) were observed in the group exposed to BPA only (Fig 6. 3). Examination of liver tissue of rats given CPF + BPA showed moderate perivascular infiltration of inflammatory cells (IC), moderate congestion (C) and mild necrosis of hepatocytes (N) (Fig.6. 4). Mild hepatocellular necrosis (N), moderate congestion (C) with mild infiltration of inflammatory cells (IC) were detected in the CPF+BPA+HES group (Fig. 6. 6). The kidney tissues of the corn oil and HES groups showed normal renal architecture (Fig. 7. 1, 5). Examination of the kidney tissues of rats given CPF only showed moderate fragmentation of the glomeruli (F) and mild congestion (C) (Fig. 7. 2). Moderate desquamation and loss of tubular epithelial cells (A) with mild focal infiltration of inflammatory cells (IC) were detected in the group exposed to BPA only (Fig. 7. 3). Examination of kidney tissue of rats co-exposed to CPF + BPA showed mild perivascular infiltration of inflammatory cells (IC) and moderate loss of tubular epithelial cells (A) (Fig. 7. 4). Mild loss of tubular epithelial cells (A) were observed in the group administered CPF+BPA+HES (Fig. 7. 6). Discussion Exposure to environmental contaminants such as pesticides and BPA at low doses poses significant health risks worldwide, as their exposure has been well-documented to inflict damage across multiple organ systems in both humans and animals. Due to their pivotal roles in the metabolism and excretion of foreign substances, the liver and kidneys have traditionally been viewed as the primary targets for various chemicals that cause toxic effects after environmental exposure. The cells of these organs are exposed to significant concentrations of chemicals, and chemical-induced hepatotoxicity and nephrotoxicity have emerged as a growing concern of global public health significance. The present study found that chronic exposure to CPF and/or BPA caused a significant increase in the activities of liver-associated enzymes, specifically AST, ALT, and ALP. Chronic co-exposure to CPF and BPA (CPF + BPA) showed the most significant elevation in AST, ALP, and ALT activity compared to exposure to either of the chemicals alone. The elevated liver enzyme activities could be linked to liver dysfunction and significant changes in the liver membrane, as evidenced by mild necrosis of the hepatocytes and moderate perivascular infiltration of inflammatory cells and congestion observed histopathologically. Previous research has reported elevated levels of liver-related enzyme activities in rats exposed to CPF alone (Zhang et al., 2021; Saoudi et al., 2021) and BPA alone (Eweda et al., 2020; Liu et al., 2022). Alanine aminotransferase is considered the most reliable and specific biomarker of hepatocellular injury, given its predominant localization within hepatocytes. Accordingly, the observed increase in ALT activity likely reflects underlying degenerative and cytotoxic changes in the liver parenchyma and associated tissues, indicative of compromised hepatocellular function. Increased serum ALP activity has been associated with pathological modifications not only in the liver but also in the bones, kidneys, intestines, and leucocytes, indicating potential multi-organ damage. The elevated ALP activity may result from oxidative damage affecting these indicated organs or direct interactions of the pesticide (Uchendu et al., 2015; Sule et al., 2022) and BPA (Liu et al., 2022). Aspartate aminotransferase is a crucial enzyme that helps turn aspartate and alpha-ketoglutarate into oxaloacetate and glutamate, playing an essential role in amino acid and energy metabolism. Accumulating evidence further indicates that BPA exposure induces hepatic injury through oxidative stress (Abdulhameed et al., 2022), with particular impact on mitochondrial integrity, alongside lipid peroxidation and inflammatory cascades. The increase observed in the activities of these enzymes may be due to oxidative damage to the liver parenchyma, as the liver is known to be centrally involved in the primary site of xenobiotic detoxification and is chronically exposed to xenobiotics and their reactive metabolites. Consequently, observed elevation in hepatic enzyme activities is likely attributable to impaired hepatocellular function and disruptions in enzyme biosynthesis, resulting in changes in the permeability of the hepatic membrane and the subsequent leakage of intracellular enzymes into the systemic circulation (Ashoush et al., 2020). Furthermore, co-exposure to CPF and BPA may have caused a release of these enzymes from the liver's cytosol into the bloodstream as a result of the pathological lesions induced in the study. Supplementation with HES attenuated the elevated hepatic enzyme activities observed in the co-exposed group, attributed to the well-documented hepato-protective properties of HES against tissue injuries. This protective effect was further corroborated histopathologically by the markedly reduced incidence and severity of pathological lesions observed in the HES-treated group. Additionally, HES have been shown to mitigate cellular damage by enhancing cellular defense mechanisms (Nasehi et al., 2023). The hepato-protective effect of HES through its ability to reduce inflammation and oxidative stress has been documented by Tabeshpour et al. (2020). The present study demonstrated that prolonged exposure to both CPF and BPA resulted in a significant elevation in serum urea concentrations. Urea is primarily filtered and excreted by the kidneys as a metabolic waste product. Therefore, the increased concentration of this metabolite after exposure to CPF and BPA may serve as a sensitive indicator of compromised renal function. Albasher et al. (2019), Afzal et al. (2022), and Sakinah et al. (2024) have shown that elevated urea concentration following CPF exposure may be a result of impaired glomerular filtration alongside tubular reabsorption. Studies have also shown that elevated urea concentration following BPA exposure may be attributed to impaired glomerular filtration and tubular function (Kobroob et al., 2018; Mohammed, 2023). The observed moderate fragmentation of the glomeruli, desquamation, and loss of tubular epithelial cells, as well as the mild congestion and focal infiltration of inflammatory cells in the rats exposed to the combination of CPF and BPA further confirm the elevated urea concentration in this study. The study demonstrates that supplementation with HES provided renal protection by significantly reducing the heightened level of urea observed in the co-exposed (CPF + BPA) group. This was also exemplified by the mild loss of tubular epithelial cells observed in the group. This finding is congruent with the works of Kucukler et al. (2021) who stated that HES reduced the high urea concentration in rats chronically exposed to CPF, and Hassan et al. (2023) who also demonstrated the protective effect of HES against aluminum-provoked renal injury in rats by reducing the increased urea concentration. The present study further demonstrated that continuous prolonged exposure to both CPF and BPA led to an increase in serum creatinine concentration. This rise in creatinine concentration provides compelling evidence of nephrotoxic injury within the renal parenchyma, as indicated by moderate fragmentation of the glomeruli, desquamation and loss of tubular epithelial cells, as well as mild congestion and focal infiltration of inflammatory cells. Previous research has also reported increased creatinine levels following exposure to CPF (Owumi and Dim, 2019; Sakinah et al., 2024) and BPA (Kobroob et al., 2018; Mohammed, 2023). Creatinine, an end-product of creatine phosphate catabolism in skeletal muscle under normal physiological conditions, is a reliable endogenous marker of glomerular filtration rate. The kidneys are responsible for removing creatinine from the body, with minimal reabsorption in the tubules. If the glomerular filtration rate decreases due to renal impairment, creatinine tends to accumulate in the bloodstream (Braun et al., 2003; Albasher et al., 2019). The rise in serum creatinine levels was more significant in the group exposed to both CPF and BPA compared to when either CPF or BPA was administered alone, suggesting that the combination caused more damage to the kidneys and/or muscles. Pretreatment with HES helped reduce the elevated creatinine concentration in the CPF + BPA group. This may be due to the anti-inflammatory and antioxidant properties of HES against CPF and BPA-induced renal and muscle damage, as evidenced by the mild loss of tubular epithelial cells observed histopathologically in this group. Hesperidin has been reported to alleviate renal damage by reducing vascular lesions, tubular cell vacuolation, and tissue damage caused by chemical contaminants (Hassan et al., 2023). Osama et al. (2024) also reported a decrease in creatinine concentration following HES supplementation in nephrotoxicity provoked by carbimazole induced hypothyroidism in adult rats. The present study demonstrated that the CPF exposure group exhibited significantly reduced total protein and albumin concentrations compared to both the co-exposed (CPF+BPA) and control group. This decrease in serum protein and albumin levels is likely due to CPF and BPA-induced hepatocellular damage, as evidenced by increased hepatic enzyme activities, mild hepatocyte necrosis, moderate congestion, and infiltration of inflammatory cells observed in the study. The observed low albumin and protein levels are consistent with previous research after exposure to CPF and Deltamethrin pesticides (Uchendu et al., 2015) and BPA (Geetharath and Josthna, 2016). Impaired hepatic albumin biosynthesis secondary to liver dysfunction or enhanced renal albumin loss through compromised tubular reabsorption, as suggested by Uchendu et al. (2015) may be responsible. Furthermore, the impact on protein integrity is more apparent than its synthesis due to BPA's induction of mitochondrial oxidative stress, leading to proteolytic degradation of existing protein pools. This is exacerbated by BPA disrupting hepatic integrity and function, collectively culminating in marked reductions (Abdel-Rahman et al., 2018). Supplementation with HES helped alleviate the decrease in serum total protein and albumin caused by CPF+BPA. This may be due to HES's antioxidative abilities, including its ability to fight off harmful free radicals and boost the body's own antioxidant defenses. Studies have shown that HES neutralizes various ROS, protecting proteins from oxidative damage (Hajialyani et al., 2019). Hesperidin has also demonstrated renoprotective attributes against oxidative damage (Khan and Parvez, 2015) by potentially reducing protein loss through renal pathways. C-reactive protein (CRP) is a pentraxin-family acute phase reactant that rises rapidly in response to tissue injury, infection, and systemic inflammation (Wu et al., 2016). Acute phase proteins, including CRP, are widely known for their diagnosis, monitoring, and prognostic assessment of a broad spectrum of acute and chronic disease conditions (Yaqub et al., 2023). The significant elevation in circulating CRP levels indicates sustained systemic inflammation and ongoing tissue injury induced by these xenobiotics. C-reactive protein can be used alongside other markers of inflammation for comprehensive assessment of both acute and chronic inflammatory burden (Yaqub et al., 2023). The CRP level was significantly increased in the CPF+BPA group compared to the individual treatment groups. Elevated C-reactive protein concentrations have been reported following CPF (Yaqub et al., 2023) and BPA (Tsen et al., 2021) exposure. The elevated concentration may be a response to specific inflammatory cytokines such as interleukin-6, likely associated with hepatocellular damage and oxidative stress induced by these contaminants in the present study. Supplementation with HES resulted in a significant drop in CRP levels. This effect may be explained by the inflammation reducing and antioxidant properties of HES. Previous studies have demonstrated that HES inhibits inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, leading to decreased prostaglandin levels and reduced inflammation. These multi-faceted mechanisms of HES likely contribute to the observed reduction in CRP concentration in the co-exposed group (Khorasanian et al., 2023). The present study showed that exposure to CPF and/or BPA increased the levels of MDA in the serum of rats. The CPF-exposed group had significantly higher MDA levels compared to the group exposed to both CPF and BPA. The observed increase in serum MDA concentration is likely due to elevated reactive oxygen species (ROS) levels and the inhibition of serum antioxidant enzyme activities identified in this study. Impairment of the enzymatic antioxidant system results in the accumulation of free radicals, which promote increased lipid peroxidation (LPO) upon exposure to environmental contaminants. Malondialdehyde, a principal product of polyunsaturated fatty acid (PUFA) peroxidation, serves as a key indicator of LPO induced by ROS in the body (Akpa et al., 2021). Chlorpyrifos and BPA, both lipophilic substances, may further enhance LPO through direct interactions with cellular membranes. The observed elevation in serum MDA concentration aligns with findings reported by other researchers following chlorpyrifos (CPF) (Kaur and Jindal, 2017) and BPA (Eweda et al., 2020) exposures. Elevated MDA concentrations following CPF exposure indicate an increase in LPO, which could potentially cause significant damage to cells and compromise the integrity of their cell membranes (Akpa et al., 2021). These reactions are closely linked to physiological functions and can act as both triggers and outcomes of cellular damage, with a well-established connection between oxidative stress and inflammation. Supplementation with HES was effective in reducing MDA levels in the CPF+BPA group. This result can be attributed to the antioxidant properties of HES, which help scavenge free radicals and increase antioxidant activities in the serum to detoxify free radicals. These findings align with those of Naseh et al. (2023) and Homayouni et al. (2017), who reported the effectiveness of HES against liver fibrosis induced by bile duct ligation in rats and Type 2 Diabetes, respectively. Although the body possesses complex inherent antioxidant defense mechanisms, exposure to xenobiotics often leads to an overproduction of reactive oxygen species (ROS) in both the intracellular and extracellular environments. This overproduction can surpass the body's natural ability to counteract oxidative damage. The present study revealed altered antioxidant enzyme activities (CAT, GPx, and SOD) in the serum of rats chronically exposed to CPF and/or BPA. The decline in SOD activity might be attributed to the direct effect of CPF and BPA or as a result of the production of free radicals induced by the contaminants. Superoxide dismutase promotes the breakdown of superoxide radicals, which are generated as consequence of heightened metabolic activity triggered by xenobiotics (Albasher et al., 2019). Superoxide dismutase also facilitates the breakdown of superoxide radicals into H 2 O 2 and O 2 . It serves as the initial enzyme involved in managing oxidative radicals (Kaur, 2017). Several authors have previously reported a reduction in SOD activity in rats exposed to CPF (Kopjar et al., 2018) and BPA (Olujimi et al., 2020). The enhanced consumption of SOD during CPF+BPA-induced autoxidation could contribute to the decline in serum SOD activity. Such reduction in SOD activity might lead to an accumulation of superoxide radicals, potentially deactivating GPx and escalating hydrogen peroxide production (Abdel-Rahman et al., 2018; Meli et al., 2020). The present study also demonstrated that exposure to CPF and/or BPA resulted in inhibition of the activity of GPx. Glutathione peroxidase is an antioxidant enzyme that contains selenium whose biological role is to protect against oxidative damage via its aiding of the conversion of hydrogen peroxide into water. The decrement in GPx activity observed in this study could potentially arise from oxidative inactivation caused by the accumulation of CPF+BPA within the body. This oxidative stress could stem from GPx inhibition, often followed by decreased levels of GSH or an elevation in hydrogen peroxide production. Several authors have previously reported a reduction in GPx activity in rats exposed to CPF (Akpa et al., 2021) and BPA (Eweda et al., 2020). Reduced GPx activity induced by CPF and BPA correlates with elevated H 2 O 2 levels and the direct suppression of SOD function. This connection underscores the impact of diminished GPx activity in raising H 2 O 2 levels within the body and concurrently inhibiting SOD's effectiveness. Taken together, these findings reveal the intricate interrelation between GPx, H 2 O 2 , and SOD in regulating oxidative processes (Eweda et al., 2020). The decline in CAT activity in this study is likely connected to the decrease induced by CPF and BPA in the activities of SOD since SOD is responsible for converting superoxide anions into hydrogen peroxide (Akpa et al., 2021). The decline in catalase activity could also result from the enzyme's depletion while trying to neutralize the hydrogen peroxide produced following co-exposure to CPF and BPA. Another factor could be the deactivation of the enzyme due to the excessive production of ROS in mitochondria and microsomes (Aboul Ezz et al., 2015). Several authors have previously reported a reduction in CAT activity in rats exposed to CPF (Albasher et al., 2019) and BPA (Aboul Ezz et al., 2015). However, the alleviation of the changes in serum antioxidant enzyme activities (SOD, GPx, and CAT) provoked by CPF+DLT exposure following HES supplementation may be due to the antioxidant attributes of HES. Hesperidin can scavenge and eliminate free radicals, effectively curtailing the buildup of free radical, and bolstering the body's inherent antioxidant defenses, possibly by amplifying the activities of endogenous antioxidant enzymes such as SOD, CAT, and GPx. This dual approach serves to protect cells from oxidative stress and potential injury (Estruel-Amades et al., 2019). Prior studies by the following authors (Pari et al. 2014; Khan and Parvez 2015; Celik et al. 2016; Caglayan et al. 2019; Nasehi et al., 2023) have consistently reported increased SOD, GPx, and CAT activities following HES supplementation in various toxicity-induced models, highlighting HES potential in counteracting oxidative stress and bolstering antioxidant defenses. In conclusion, exposure of rats to low doses of both CPF and BPA over a period of time, caused more alterations in certain serum biochemical parameters (such as AST, ALP, ALT activities, and urea, creatinine, and CRP levels) compared to those exposed to just one of the contaminants. It can be inferred that the co-exposure to CPF and BPA resulted in more negative effects by triggering inflammation and oxidative stress. The use of HES as a supplement helped improve the impaired kidney and liver functions, partly by reducing urea, creatinine, and CRP levels, as well as AST, ALT, and ALP activities. Additionally, it helped mitigate oxidative stress by decreasing LPO, thereby restoring the rats' antioxidant status. Abbreviations ALP Alkaline phosphatase ALT Alanine aminotransferase ANOVA One-way analysis of variance AST Aspartate aminotransferase BPA Bisphenol A CAT Catalase CPF Chlorpyrifos CRP C-reactive proteins DLT Deltamethrin GPx Glutathione peroxidase HE Hematoxylin eosin HES Hesperidin IL-1 Interleukin-1 iNOS inducible nitric oxide synthase LD50 Median lethal dose 50 LOAEL Lowest observed adverse effect level LPO Lipid peroxidation MDA Malondialdehyde ROS Reactive oxygen species SEM Standard error of the mean SOD Superoxide dismutase TBA Thiobarbituric acid Declarations Acknowledgements: Not applicable Ethics approval and consent to participate Ethical approval was sort for use of animals from the Animal Care and Use Committee of Animal Experimental Unit of the Department of Pharmacology, University of Jos, Nigeria. Consent of publication Not applicable. Data Availability All data and materials are available on reasonable request. Competing Interests The authors have declared that no competing interests exist. Conflict of interest The authors declare that they have no conflict of interest. Funding sources The authors received no specific funding for this work. Author’s Contributions Names of Authors Contributions of Authors Enokela Shaibu IDOGA Implementation of the research. Nendirmwa Musa DASHE Design of the work, implementation of the research and drafting of manuscript. Onuche Shalom AGWECHE Implementation of the research. Blessing EDOGBO Drafting of manuscript and review. Joy Iyojo ITODO. Drafting of manuscript and review. Chidiebere UCHENDU Design of the work, implementation of the research, data analysis and interpretation, writing of manuscript and review. References Abdel-Rahman, H.G., Abdelrazek, H.M.A., Zeidan, D.W., Mohamed, R.M. & Abdelazim, A.M. (2018). Lycopene: hepatoprotective and antioxidant effects toward bisphenol A-induced toxicity in female Wistar rats . Oxidative Medicine and Cellular Longevity, doi: 10.1155/2018/5167524. Abdulhameed, A.A.R., Lim, V., Bahari, H., Khoo, B.Y., Abdullah, M.N.H., Tan, J.J. &Yong, Y.K. (2022). Adverse effects of bisphenol A on the liver and its underlying mechanisms: evidence from in-vivo and in-vitro studies . Biomed Research International, doi: 10.1155/2022/8227314. Aboul Ezz, H.S., Khadrawy, Y.A. & Mourad, I.M. (2015). The effect of bisphenol A on some oxidative stress parameters and acetylcholinesterase activity in the heart of male albino rats. Cytotechnology, 67,145-155. doi: 10.1007/s10616-013-9672-1. Adiga, D., Nadeem Khan, G., Eswaran, S., Sriharikrishnaa, S., Chakrabarty, S., Rai, P.S. & Kabekkodu, S.P. (2022). Bisphenol A associated signaling pathways in human diseases. In: Gassman NR (ed) Bisphenol A: a multi-modal endocrine disruptor. Royal Society of Chemistry Cambridge, pp 42–86. https://doi.org/10.1039/9781839166495-00042. Akpa, A.R., Ayo, J.O., Mika’il, H.G. & Zakari, F.O. (2021). Protective effect of fisetin against subchronic chlorpyrifos-induced toxicity on oxidative stress biomarkers and neurobehavioral parameters in adult male albino mice. Toxicological Research, 37, 163-171. doi.org/10.1007/s43188-020-00049-y. Albasher, G., Almeer, R., Alarifi, S., Alkhtani, S., Farhood, M., Al-Otibi, F.O. & Rizwana, H. (2019). Nephroprotective role of Beta vulgaris L . root extract against chlorpyrifos-induced renal injury in rats. Evidence Based Complementary and Alternative Medicine, doi: 10.1155/2019/3595761. Amjad, S., Rahman, M.S. & Pang, M.G. (2020). Role of antioxidants in alleviating bisphenol A toxicity. Biomolecules, 10(8), 1105. doi: 10.3390/biom10081105. Ashoush, Y., Abozid, M., Mansour, S. & Morgan, A. (2020). Effect of chlorpyrifos on liver function of albino rates. Menoufia Journal of Agricultural Biotechnology, 5(2), 83-92. doi: 10.21608/MJAB.2020.170415. Bala, R., Dhingra, S., Kumar, M., Bansal, K., Mittal, S., Sharma, R.K. & Wangoo, N. (2017). Detection of organophosphorus pesticide–Malathion in environmental samples using peptide and aptamer based nanoprobes. Chemical Engineering Journal, 311, 111-116. doi.10.1016/j.cej.2016.11.070. Beers, R.F. & Sizer, I.W. (1952). A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. Journal of Biological Chemistry, 195(1), 133-140. Bloch, D., Diel, P., Epe, B., Hellwig, M., Lampen, A., Mally, A., Marko, D., Villar Fernández, M.A., Guth, S., Roth, A., Marchan, R., Ghallab, A., Cadenas, C., Nell, P., Vartak, N., van Thriel, C., Luch, A., Schmeisser, S., Herzler, M., Landsiedel, R., Leist, M., Marx‑Stoelting, P., Tralau, T. & Hengstler, J.G. (2023). Basic concepts of mixture toxicity and relevance for risk evaluation and regulation. Archives of Toxicology, 97, 3005–3017. https://doi.org/10.1007/s00204-023-03565-6. Braun, J.P., Lefebvre, H.P. & Watson, A.D.J. (2003). Creatinine in the dog: a review. Veterinary Clinical Pathology, 32(4), 162-179. doi: 10.1111/j.1939-165x.2003.tb00332.x. Caglayan, C., Demir, Y., Kucukler, S., Taslimi, P., Kandemir, F.M. & Gulçin, İ. (2019). The effects of hesperidin on sodium arsenite‐induced different organ toxicity in rats on metabolic enzymes as antidiabetic and anticholinergics potentials: A biochemical approach. Journal of Food Biochemistry, 43(2), e12720. doi: 10.1111/jfbc.12720. Celik, E., Oguzturk, H., Sahin, N., Turtay, M.G., Oguz, F. & Ciftci, O. (2016). Protective effects of hesperidin in experimental testicular ischemia/reperfusion injury in rats. Archives of Medical Science, 12(5), 928-934. doi: 10.5114/aoms.2015.47697. Choi, C.W., Jeong, J.Y., Hwang, M.S., Jung, K.K., Lee, K.H. & Lee, H.M. (2010). Establishment of the Korean tolerable daily intake of bisphenol A based on risk assessments by an expert committee. Toxicological Research, 26(4), 285-291. doi.org/10.5487/TR.2010.26.4.285. Coppola, L., Lori, G., Talt, S., Sogorb, M.A. & Estevan, C. (2025). Evaluation of developmental toxicity of chlorpyrifos through new approach methodologies: a systematic review. Archives of Toxicology , 99, 935–981. doi.org/10.1007/s00204-024-03945-6. Costa, H.E., Medeiros, I., Mariana, M. & Cairrao. E. (2025). Maternal–foetal effects of exposure to Bisphenol A: outcomes and long-term consequence. Applied Sciences , 15,697. https://doi.org/10.3390/ app15020697. das Neves, R.N., Carvalho, F., Carvalho, M., Fernandes, E., Soares, E., de Bastos, M.L. & de Pereira, M.L. (2004). Protective activity of hesperidin and lipoic acid against sodium arsenite acute toxicity in mice. Toxicologic Pathology, 32(5), 527-35. doi: 10.1080/01926230490502566. Draper, H.H. & Hadley, M. (1990). Malondialdehyde determination as index of lipid Peroxidation . In Methods in Enzymology, 186, 421-431. El-Gameel, D., Hamdy, N.A., El-Yazbi, A.F., Ghanem, M.A., El-Khordaugi, L.K., Abdallah, S.M., Mechref, Y. & El-Yazbi, A.F. (2024). Chronic exposure to organophosphate pesticides and elevated markers of systemic inflammation: Possible neuroinflammatory and genotoxic effects. Journal of Pharmacology and Experimental Therapeutics, 385 (S3) 27. doi.org/10.1124/jpet.122.190690. Estruel-Amades, S., Massot-Cladera, M., Garcia-Cerdà, P., Pérez-Cano, F.J., Franch, À. Castell, M. & Camps-Bossacoma, M. (2019). Protective effect of hesperidin on the oxidative stress induced by an exhausting exercise in intensively trained rats. Nutrients, 11(4), 783.doi: 10.3390/nu11040783. Eweda, S.M., Newairy, A.S.A., Abdou, H.M. & Gaber, A.S. (2020). Bisphenol A‑induced oxidative damage in the hepatic and cardiac tissues of rats: The modulatory role of sesame lignans. Experimental and Therapeutic Medicine, 19(1), 33-44. doi: 10.3892/etm.2019.8193. Geetharathan, T. & Josthna, P. (2016). Effect of BPA on protein, lipid profile and immuno-histo chemical changes in placenta and uterine tissues of albino rat . International Journal of Pharmaceutical and Clinical Research, 8(4), 260-268. Grandjean, P. & Landrigan, P.J. (2014). Neurobehavioural effects of developmental toxicity. The Lancet Neurology, 13(3), 330-338. doi: 10.1016/S1474-4422(13)70278-3. Hajialyani, M., Hosein Farzaei, M., Echeverría, J., Nabavi, S.M., Uriarte, E. & Sobarzo-Sánchez, E. (2019). Hesperidin as a neuroprotective agent: a review of animal and clinical evidence. Molecules, 24(3), 648. doi: 10.3390/molecules24030648. Hassan, Z.K., Elobeid, M.A., Virk, P., Omer, S.A., Elamin, M., Daghestani, M.H. & Alolayan, E.M. (2012). Bisphenol A induces hepatotoxicity through oxidative stress in rat model. Oxidative Medicine and Cellular Longevity, doi: 10.1155/2012/194829. Hassan, N.H., Yousef, D.M. & Alsemeh, A.E. (2023). Hesperidin protects against aluminum-induced renal injury in rats via modulating MMP-9 and apoptosis: biochemical, histological, and ultrastructural study . Environmental Science and Pollution Research International, 30(13), 36208-36227. doi: 10.1007/s11356-022-24800-0. Helal, E.G.E., Badawi, M.M.M., Soliman, M.G., Abdel-Kawi, N.A., Fadel, H.A.E. & Abozaid, N.M.G. (2013). Physiological and histopathological studies on bisphenol-A compound as xenoestrogen in male albino rats. The Egyptian Journal of Hospital Medicine, 50, 127 – 136.doi. 10.21608/EJHM.2018.16081. Homayouni, F., Haidari, F., Hedayati, M., Zakerkish, M. & Ahmadi, K. (2017). Hesperidin supplementation alleviates oxidative DNA damage and lipid peroxidation in type 2 diabetes: A randomized double‐blind placebo‐controlled clinical trial. Phytotherapy Research, 31(10), 1539-1545. doi: 10.1002/ptr.5881. Katiyar, D., Sexena, R., Kumar, A., Bansal, P., Prakash, S., Ghosh, D. & Nagarajan, K. (2024). A comprehensive review of the protective effects of herbals against toxicity of Bisphenol A. Toxin Reviews , 43(3), 329-357. doi.org/10.1080/15569543.2024.2329907. Kaur, M. & Jindal, R. (2017). Oxidative stress response in liver, kidney and gills of Ctenopharyngodon idellus (cuvier & valenciennes) exposed to chlorpyrifos. MOJ Biology and Medicine, 1(4), 103-112. doi.10.15406/mojbm.2017.01.00021. Khan, M.H.A. & Parvez, S. (2015). Hesperidin ameliorates heavy metal induced toxicity mediated by oxidative stress in brain of Wistar rats. Journal of Trace Elements in Medicine and Biology, 31, 53-60. doi: 10.1016/j.jtemb.2015.03.002. Khorasanian, A.S., Fateh, S.T., Gholami, F., Rasaei, N., Gerami, H., Khayyatzadeh, S.S. & Asbaghi, O. (2023). The effects of hesperidin supplementation on cardiovascular risk factors in adults: a systematic review and dose–response meta-analysis. Frontier in Nutrition, 10, 1177708. doi: 10.3389/fnut.2023.1177708. Kobroob, A., Peerapanyasut, W., Chattipakorn N. & Wongmekiat, O. (2018). Damaging effects of bisphenol A on the kidney and the protection by melatonin: Emerging evidences from in vivo and in vitro studies. Oxidative Medicine and Cellular Longevity, doi: 10.1155/2018/3082438. Konieczna, A., Rutkowska, A. & Rachoń, D. (2015). Health risk of exposure to bisphenol A (BPA). Rocz Panstw Zakl Hig, 66(1), 5-11. Kopjar, N., Žunec, S., Mendaš, G., Micek, V., Kašuba, V., Mikolić, A. & Želježić, D. (2018). Evaluation of chlorpyrifos toxicity through a 28-day study: Cholinesterase activity, oxidative stress responses, parent compound/metabolite levels, and primary DNA damage in blood and brain tissue of adult male Wistar rats. Chemico- Biological Interaction s, 279, 51-63. doi: 10.1016/j.cbi.2017.10.029. Küçükler, S., Çomaklı, S., Özdemir, S., Çağlayan, C. & Kandemir, F.M. (2021). Hesperidin protects against the chlorpyrifos-induced chronic hepato-renal toxicity in rats associated with oxidative stress, inflammation, apoptosis, autophagy, and up-regulation of PARP-1/VEGF. Environmental Toxicology, 36(8), 1600-1617. https://doi.org/10.1002/tox.23156. Liguori, I., Russo, G., Curcio, F., Bulli, G., Aran, L., Della-Morte, D. & Abete, P. (2018). Oxidative stress, aging, and diseases. Clinical Intervention in Aging, 13, 757-772. doi: 10.2147/CIA.S158513. Liu, T.Y., Wang, C., Han, Y.Z., Bai, C., Ren, H.T., Liu, Y. & Han, X. (2022). Oxidative polymerization of bisphenol A (BPA) via H-abstraction by Bi2.15WO6 and persulfate: Importance of the surface complexes. Chemical Engineering Journal, l 435, 134816. doi: 10.1016/j.cej.2022.134816. Luna LG. (1968). Manual of histologic staining methods of the Armed Forces Institute of Pathology. In Manual of histologic staining methods of the Armed Forces Institute of Pathology (pp. xii-258). Manzetti, S., van der Spoel, E.R. & van der Spoel, D. (2014). Chemical properties, environmental fate, and degradation of seven classes of pollutants. Chemical Research in Toxicology 27(5):713-37. doi: 10.1021/tx500014w. Martin Jr J. P., Dailey, M. & Sugarman, E. (1987). Negative and positive assays of superoxide dismutase based on hematoxylin autoxidation. Archives of Biochemistry and Biophysics, 255(2), 329-36. doi: 10.1016/0003-9861(87)90400-0. Meli, R., Monnolo, A., Annunziata, C., Pirozzi, C. & Ferrante, M.C. (2020). Oxidative stress and BPA toxicity: an antioxidant approach for male and female reproductive dysfunction. Antioxidants, 9(5), 405. https://doi.org/10.3390/antiox9050405. Mohammed, A. (2023). Lycopene attenuates oxidative stress, apoptosis and biochemical fluctuations induced by bisphenol A in the kidneys of rats. European Journal of Anatomy, 27 (5), 529-540. Nasehi, Z., Kheiripour, N., Taheri, M.A., Ardjmand, A., Jozi, F. & Shahaboddin, M.E. (2023). Efficiency of Hesperidin against liver fibrosis Induced by bile duct ligation in rats . Biomed Research International, doi : 10.1155/2023/5444301 . National Research Council (1996) Guide for the care and use of laboratory animals. Academic Press, Washington. Nayak, D., Adiga, D., Khan, N.G., Rai, P.S., Dsouza, H.S., Chakrabarty, S. & Gassman, N.R., Kabekkodu, S.P. (2022). Impact of bisphenol A on structure and function of mitochondria: A Critical Review. Reviews Environmental Contamination (formerly: Residue Reviews), 260, 10. doi.org/10.1007/s44169-022-00011-z. Noshy, P.A., Khalaf, A.A.A., Ibrahim, M.A., Mekkawy, A.M., Abdelrahman,, R.E., Farghali, A., Tammam, A.A. & Zaki, A.R. (2022). Alterations in reproductive parameters and steroid biosynthesis induced by nickel oxide nanoparticles in male rats. The ameliorative effect of hesperidin. Toxicology, 473, 153208.doi.10.1016/j.tox.2022.153208. Olujimi, O., Ayoola, R., Olayinka, O., Dosumu, O., Rotimi, S. & Aladesida, A. (2020). Evaluation of antioxidant enzymes performances and DNA damage induced by bisphenol A and diisobutylphthalate in Hyperiodrilus africanus -earthworms. Emerging Contaminants, 6, 1-9. doi.10.1016/j.emcon.2019.10.001. Omodon, A.C., Onwuka, O.M., Adele, B.O. & Ige, A.O. (2024). cardiotoxic effects of Bisphenol A in male wistar rats are attenuated by Garcinia kola and its biflavonoid, kolaviron, via antioxidant and antiinflammation-based mechanisms. Journal of Traditional and Complementary Medicine , doi.org/10.1016/j.jtcme.2024.08.004. Osama, H.M., Khadrawy, S.M., El-Nahass, E., Othman, S.I. & Mohamed, H.M. (2024). Eltroxin and Hesperidin mitigate testicular and renal damage in hypothyroid rats: amelioration of oxidative stress through PPARγ and Nrf2/HO-1 signaling pathway. Laboratory Animal Research , 40, 19. doi.org/10.1186/s42826-024-00204-8. Owumi, S.E. & Dim, U.J. (2019). Manganese suppresses oxidative stress, inflammation and caspase-3 activation in rats exposed to chlorpyrifos. Toxicology Reports, 6, 202-209. doi: 10.1016/j.toxrep.2019.02.007. Paglia, D.E. & Valentine, W.N. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. Journal of Laboratory and Clinical Medicine, 70(1), 158-169. Pari, L., Karthikeyan, A., Karthika, P. & Rathinam, A. (2015). Protective effects of hesperidin on oxidative stress, dyslipidaemia and histological changes in iron-induced hepatic and renal toxicity in rats. Toxicology Reports, 2, 46-55. doi: 10.1016/j.toxrep.2014.11.003. Pelletier, G. Wang, G.S., Wawrzynczak, A., Rigden, M., Aranda-Rodriguez, R. & Caldwell, D. (2026). Direct Comparison of the Impacts of Bisphenol A, Bisphenol F, and Bisphenol S in a Male Rat 28-Day Oral Exposure Study. International Journal of Toxicology, 45(1), 4–21. doi: 10.1177/10915818251378990. Pyrzynska, K. (2022). Hesperidin: A review on extraction methods, stability and biological activities. Nutrients, 14(12), 2387. https://doi.org/10.3390/nu14122387. Rather, I.A., Koh, W.Y., Paek, W.K. & Lim, J. (2017). The sources of chemical contaminants in food and their health implications. Frontier in Pharmacology, 17, 8:830. doi: 10.3389/fphar.2017.00830. Sakinah, E.N., Wisudanti, D.D., Abrori, C., Supangat, S., Ramadhani, L.R., Putri, I.S., Pamungkas, G.C., Arrobani, M.H., Rahmadina, R. & Dirgantara, W. (2024). The effect of chlorpyrifos oral exposure on the histomorphometric and kidney function in Wistar rat. Indian Journal Pharmacology , 56, 186-90. Saoudi, M., Badraoui, R., Rahmouni, F., Jamoussi, K. & El Feki, A. (2021). Antioxidant and protective effects of artemisia campestris essential oil against chlorpyrifos-induced kidney and liver injuries in rats. Frontier in Physiology, 12 – 2021. https://doi.org/10.3389/fphys.2021.618582. Steffensen, I.L., Dirven, H., Couderq, S., David, A., D'Cruz, S.C., Fernández, M.F., Mustieles, V., Rodríguez-Carrillo, A. & Hofer, T. (2020). Bisphenols and oxidative stress biomarkers-associations found in human studies, evaluation of methods used, and strengths and weaknesses of the biomarkers. International Journal of Environmental Research and Public Health, 17(10), 3609. doi: 10.3390/ijerph17103609. Sule, R.O., Condon, L. & Gomes, A.V. (2022). A common feature of pesticides: oxidative stress—the role of oxidative stress in pesticide-induced toxicity. Oxidative Medicine and Cellular Longevity, doi: 10.1155/2022/5563759. Tabeshpour, J., Hosseinzadeh, H., Hashemzaei, M. & Karimi, G. (2020). A review of the hepatoprotective effects of hesperidin, a flavanon glycoside in citrus fruits, against natural and chemical toxicities. Daru Journal of Pharmaceutical Sciences, 28(1), 305-317. doi: 10.1007/s40199-020-00344-x. Tsen, C.M., Liu, J.H., Yang, D.P., Chao, H.R., Chen, J.L., Chou, W.C. & Chuang, C.Y. (2021). Study on the correlation of bisphenol A exposure, pro-inflammatory gene expression, and C-reactive protein with potential cardiovascular disease symptoms in young adults. Environmental Science and Pollution Research International, doi: 10.1007/s11356-021-12805-0. Uchendu, C., Ambali, S.F., Ayo, J.O. & Esievo, K.A.N. (2015). The protective role of alpha-lipoic acid on long-term exposure of rats to the combination of chlorpyrifos and deltamethrin pesticides. Toxicology and Industrial Health, 31(12), 1061-1347.doi.org/10.1177/0748233715616553. Uchendu, C., Ambali, S.F., Ayo, J.O., Esievo, K.A.N. & Umosen, A.J. (2014). Erythrocyte osmotic fragility and lipid peroxidation following chronic co-exposure of rats to chlorpyrifos and deltamethrin, and the beneficial effect of alpha-lipoic acid. Toxicology Reports, 1, 373-378. doi.org/10.1016/j.toxrep.2014.07.002. Vandenberg, L.N., Ehrlich, S., Belcher, S.M., Ben-Jonathan, N., Dolinoy, D.C., Hugo, E.R., Hunt, P.A., Newbold, R.R., Rubin, B.S., Sail, K.S., Soto, A.M., Wang, H. & vom Saal, F.S. (2013). Low dose effects of bisphenol A an integrated review of in vitro, laboratory animal, and epidemiology studies. Endocrine Disruptors, 1, 1, e25078. doi: 10.4161/endo.26490. Wołejko, E., Łozowicka, B., Jabłońska-Trypuć, A., Pietruszyńska, M. & Wydro, U. (2022). Chlorpyrifos occurrence and toxicological risk assessment: a review. International Journal of Environmental Research and Public Health, 19 (19), 12209. doi: 10.3390/ijerph191912209. Wu, X., Xie, W., Cheng, Y. & Guan, Q. (2016). Severity and prognosis of acute organophosphorus pesticide poisoning are indicated by C-reactive protein and copeptin levels and APACHE II score. Experimental and Therapeutic Medicine, 11(3), 806-810. doi: 10.3892/etm.2016.2982. Wu, Y., Chang, S., Chen, H., Tsai, K., Lee, W., Wang, I., Lee, C., Chen, C., Liu, S., Weng, C., Huang, W., Hsu, C. & Yen, T. (2023). Human poisoning with chlorpyrifos and cypermethrin pesticide mixture: Assessment of clinical outcome of cases admitted in a tertiary care hospital in Taiwan. International Journal of General Medicine, 16, 4795-4804. doi: 10.2147/IJGM.S432861. Yaqub, S.A., Rahamon, S.K. & Arinola, O.G. (2023). Serum levels of selected inflammatory markers in farm workers exposed to organophosphate pesticides. African Journal of Biomedical Research 26(1): 89-93. Yavuz, T., Delibas, N., Yildirim, B., Altuntas, I., Candır, O., Cora, A. & Kutsal, A. (2004). Vascular wall damage in rats induced by methidathion and ameliorating effect of vitamins E and C. Archives of Toxicology, 78(11), 655-9. doi: 10.1007/s00204-004-0593-9. Zhang, Y., Jia, Q., Hu, C., Han, M., Guo, Q., Li, S. & Peng, C. (2021). Effects of chlorpyrifos exposure on liver inflammation and intestinal flora structure in mice. Toxicology Research (Camb), 10(1), 141-149. doi: 10.1093/toxres/tfaa108. Table Table 1: Effect of chronic exposure to corn oil (C/oil), chlorpyrifos (CPF), bisphenol A (BPA), chlorpyrifos and bisphenol A (CPF+BPA), hesperidin (HES) and chlorpyrifos, bisphenol A and hesperidin (CPF+BPA+HES) on serum oxidative stress biomarkers in Wistar rats. Parameters C/oil CPF BPA CPF+BPA HES CPF+BPA+HES SOD (IUL -1 ) 3.55±0.15 2.05±0.18 a 2.55±0.05 2.18±0.33 a 4.37±0.13 bcd 3.2±0.15 b CAT (IUL -1 ) 9.83±0.4 2.68±0.66 a 5.8±0.40 ab 2.75±0.61 ac 8.8±0.62 bcd 6.03±0.56 abde GPx (IUL -1 ) 1.17±0.03 0.36±0.05 a 0.9±0.10 0.68±0.12 1.53±0.32 bd 1.1±0.24 b MDA (nmolml -1 ) 3.9±0.53 10.02±1.00 a 5.1±0.23 b 6.17±0.87 b 2.6±0.29 b 4.16±0.68 bd a P < 0.05 compared to C/oil group b P < 0.05 compared to CPF group c P< 0.05 compared to BPA group d P < 0.05 compared to CPF+BPA group e P < 0.05 compared to HES group Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9163020","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":635402405,"identity":"72d651f4-e427-4850-a5f4-45d945534c0e","order_by":0,"name":"Enokela Shaibu Idoga","email":"","orcid":"","institution":"University of Jos","correspondingAuthor":false,"prefix":"","firstName":"Enokela","middleName":"Shaibu","lastName":"Idoga","suffix":""},{"id":635402406,"identity":"2a3c0007-18f4-49b0-ba0f-06ec6f52feed","order_by":1,"name":"Nendirmwa Musa Dashe","email":"","orcid":"","institution":"University of Jos","correspondingAuthor":false,"prefix":"","firstName":"Nendirmwa","middleName":"Musa","lastName":"Dashe","suffix":""},{"id":635402407,"identity":"e85d793e-4249-4506-9a77-a3e5b884b1d1","order_by":2,"name":"Onuche Shalom Agweche","email":"","orcid":"","institution":"University of Jos","correspondingAuthor":false,"prefix":"","firstName":"Onuche","middleName":"Shalom","lastName":"Agweche","suffix":""},{"id":635402408,"identity":"cd6b078f-894b-4517-bfe0-45f1ec8a51e0","order_by":3,"name":"Blessing Edogbo","email":"","orcid":"","institution":"National Open University, Abuja, Nigeria.","correspondingAuthor":false,"prefix":"","firstName":"Blessing","middleName":"","lastName":"Edogbo","suffix":""},{"id":635402409,"identity":"1b257f15-6ab4-4687-827f-75e463b557cf","order_by":4,"name":"Joy Iyojo Itodo","email":"","orcid":"","institution":"Federal University Lafia","correspondingAuthor":false,"prefix":"","firstName":"Joy","middleName":"Iyojo","lastName":"Itodo","suffix":""},{"id":635402410,"identity":"28ee7495-4ee7-49d8-bf3e-1c801be1c5ed","order_by":5,"name":"Chidiebere Uchendu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYDACCcYGiQQGhgR+BgY2ErVINhCvBYwYEgwOEKuFf3Zz442HbXZ5xjeSnz34UMEgzy92gIAldw42WyS2JReb3UgzN5xxhsFw5uwE/FoMJBLbgOhA4rYbCWbSvG1AF94mVsvmGenfSNSyQSKHSFskbiQ2WyScS06cceZNmeSMMxKE/cI/I/3hzR9ldon97enbJD5U2MjzSxPQggACYJUSxCoH23eAFNWjYBSMglEwkgAADUJE+HbvzyIAAAAASUVORK5CYII=","orcid":"","institution":"University of Jos","correspondingAuthor":true,"prefix":"","firstName":"Chidiebere","middleName":"","lastName":"Uchendu","suffix":""}],"badges":[],"createdAt":"2026-03-18 21:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9163020/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9163020/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108709240,"identity":"4b720678-b9e4-4046-a627-2e6195a3eaa2","added_by":"auto","created_at":"2026-05-07 13:57:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50327,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of chronic exposure to corn oil (C/oil), chlorpyrifos (CPF), bisphenol A (BPA), chlorpyrifos and bisphenol A (CPF+BPA), hesperidin (HES) and chlorpyrifos, bisphenol A and hesperidin (CPF+BPA+HES) on liver function enzymes activities in Wistar rats (n =5). \u003csup\u003ea\u003c/sup\u003eP \u0026lt; 0.05 versus CPF, BPA and CPF+BPA groups, respectively; \u003csup\u003eb\u003c/sup\u003eP \u0026lt; 0.05 versus HES and CPF+BPA+HES groups, respectively; \u003csup\u003ec\u003c/sup\u003eP \u0026lt; 0.05 versus BPA and CPF+BPA groups, respectively; \u003csup\u003ed\u003c/sup\u003eP \u0026lt; 0.05 versus CPF+BPA group.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9163020/v1/0a4782222caf04bfbba59b32.png"},{"id":108709252,"identity":"1079424f-b234-45ad-8d8d-e078abb65422","added_by":"auto","created_at":"2026-05-07 13:57:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":21072,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of chronic exposure to corn oil (C/oil), chlorpyrifos (CPF), bisphenol A (BPA), chlorpyrifos and bisphenol A (CPF+BPA), hesperidin (HES) and chlorpyrifos, bisphenol A and hesperidin (CPF+BPA+HES) on serum urea concentration in Wistar rats (n= 5). \u003csup\u003ea\u003c/sup\u003eP \u0026lt; 0.05 versus C/oil, CPF, BPA, HES and CPF+BPA+HES groups, respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9163020/v1/d612d8037374a02187dd7c90.png"},{"id":108709230,"identity":"c8a42a9e-25b2-4a41-b1a3-6e5e6123ee10","added_by":"auto","created_at":"2026-05-07 13:57:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21941,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of chronic exposure to corn oil (C/oil), chlorpyrifos (CPF), bisphenol A (BPA), chlorpyrifos and bisphenol A (CPF+BPA), hesperidin (HES) and chlorpyrifos, bisphenol A and hesperidin (CPF+BPA+HES) on serum creatinine concentration in Wistar rats (n =5). P \u0026lt; 0.05 versus C/oil, BPA, HES and CPF+BPA+HES groups, respectively.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9163020/v1/4c9d80f325de861fce7b7f8f.png"},{"id":108709239,"identity":"539ae623-4117-4903-b473-30615957265b","added_by":"auto","created_at":"2026-05-07 13:57:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":66655,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of chronic exposure to corn oil (C/oil), chlorpyrifos (CPF), bisphenol A (BPA), chlorpyrifos and bisphenol A (CPF+BPA), hesperidin (HES) and chlorpyrifos, bisphenol A and hesperidin (CPF+BPA+HES) on serum proteins concentration in Wistar rats (n =5). \u003csup\u003ea\u003c/sup\u003eP \u0026lt; 0.05 versus C/oil and HES groups, respectively; \u003csup\u003eb\u003c/sup\u003eP \u0026lt; 0.05 versus CPF and CPF+BPA groups, respectively; \u003csup\u003ec\u003c/sup\u003eP \u0026lt; 0.05 versus C/oil, BPA, HES and CPF+BPA+HES groups, respectively.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9163020/v1/e7c11e1fec9134f6fc8c8cce.png"},{"id":108709251,"identity":"26a3ec58-3025-438e-bae8-e33eb511263c","added_by":"auto","created_at":"2026-05-07 13:57:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":21701,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of chronic exposure to corn oil (C/oil), chlorpyrifos (CPF), bisphenol A (BPA), chlorpyrifos and bisphenol A (CPF+BPA), hesperidin (HES) and chlorpyrifos, bisphenol A and hesperidin (CPF+BPA+HES) on C reactive protein concentration in Wistar rats (n =5). aP \u0026lt; 0.05 versus CPF, BPA and CPF+BPA groups, respectively; \u003csup\u003eb\u003c/sup\u003eP \u0026lt; 0.05 versus HES and CPF+BPA+HES groups, respectively; \u003csup\u003ec\u003c/sup\u003eP \u0026lt; 0.05 versus BPA, CPF+BPA and CPF+BPA+HES groups, respectively; P \u0026lt; 0.05 versus HES and CPF+BPA+HES groups, respectively.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9163020/v1/acd3d72dd7a457c2e96a0a22.png"},{"id":108709232,"identity":"5b9c8778-0a3c-405e-a259-5959224b4b59","added_by":"auto","created_at":"2026-05-07 13:57:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1772329,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs analysis of control (C/oil), the Chlorpyrifos only (CPF), the bisphenol A only (BPA), the combination of Chlorpyrifos and bisphenol A (CPF+BPA), the hesperidin only (HES) and the combinations of chlorpyrifos, bisphenol A and hesperidin (CPF+BPA+HES) in rat liver stained with haematoxylin and eosin viewed at original magnification (x400). (1) normal hepatocellular architecture indicating normal hepatocytes; (2) mild loss of hepatocytes (L) and mild focal infiltration of inflammatory cells (IC); (3) moderate congestion (C), mild loss of hepatocytes (L) and moderate accumulation of eosinophilic material in the hepatic vein (E); (4) moderate perivascular infiltration of inflammatory cells(IC), moderate congestion(C) and mild necrosis of hepatocytes(N); (5) normal hepatocellular architecture; (6) mild hepatocellular necrosis (N), moderate congestion(C) with mild infiltration of inflammatory cells(IC).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9163020/v1/8a983759bb0a1df99be65573.png"},{"id":108709241,"identity":"dde61124-cd84-4606-bbe6-12f3350953c7","added_by":"auto","created_at":"2026-05-07 13:57:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1657975,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs analysis of control (C/oil), the Chlorpyrifos only (CPF), the bisphenol A only (BPA), the combination of Chlorpyrifos and bisphenol A (CPF+BPA), the hesperidin only (HES) and the combinations of chlorpyrifos, bisphenol A and hesperidin (CPF+BPA+HES) in rat kidneys stained with haematoxylin and eosin viewed at original magnification (x400). (1) normal renal architecture; (2) moderate fragmentation of the glomeruli (F) and mild congestion(C); (3) moderate desquamation and loss of tubular epithelial cells (A) with mild focal infiltration of inflammatory cells (IC); (4) mild perivascular infiltration of inflammatory cells (IC) and moderate loss of tubular epithelial cells (A); (5) normal renal morphology; (6) mild loss of tubular epithelial cells (A).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9163020/v1/1d927d8c4a73f4400dcb56fe.png"},{"id":108709338,"identity":"81aca930-da7d-4c95-b2df-b9c38bfb802f","added_by":"auto","created_at":"2026-05-07 13:57:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5813558,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9163020/v1/861eca35-ebbe-4ef9-97fa-8dd81d8247f1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chronic Hepatorenal Toxicity Induced by Low-Dose Co-Exposure to Chlorpyrifos and Bisphenol A in Wistar Rats: Protective Effects of Hesperidin","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u0026nbsp;\u0026nbsp;Environmental contaminants from industrialization pose significant challenges to public health and the environment as they are capable of causing harm to humans, animals, and ecosystems (Grandjean and Landrigan, 2014). These contaminants encompass a variety of substances including pesticides, heavy metals, radioactive elements, and endocrine disruptors like bisphenol A (Manzetti et al., 2014).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; Bisphenol A (BPA) is a hazardous chemical that is extensively produced, widely utilized as a synthetic product worldwide and is commonly found in food packaging, medical devices, feeding bottles, and various plastic items (Katiyar et al., 2024; Omodon et al., 2024;\u0026nbsp;Pelletier et al., 2026). The preponderate use of BPA has the capacity to cause damage to humans, animals and the environment. Exposure to BPA occurs chiefly via ingestion, inhalation, and transdermal routes (Nayak et al., 2022). Among these, the consumption of BPA-contaminated food, water, and beverages represents the most significant pathway of entry into the body (Helal et al., 2013; Begum et al., 2020; Adiga et al., 2022). Given BPA\u0026rsquo;s high global production volume and its tendency to leach from plastic products, exposure among both humans and animals has become pervasive (Steffenson et al., 2020). \u0026nbsp;Among all endocrine disruptors, BPA is one of the most manufactured synthetic substances globally (Costa et al., 2025). \u0026nbsp;Bisphenol A has emerged as a compound of significant public health concern, because people can be exposed to it in many ways, and even small amounts can cause harm (Konieczna et al., 2015).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;Organophosphates (OPs) represents one of the most extensively used classes of pesticides, accounting for approximately 50% of all pesticides used globally (Wołejko et al., 2022). The persistent presence of OP residues in the environment poses significant public health concerns worldwide as these residues can be found in food and water. Developing nations, such as Nigeria, bear a disproportionately higher burden of pesticide poisoning due to unregulated exposure from consumption of contaminated food and water (Bala et al., 2017).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Chlorpyrifos (CPF) is an OP insecticide with wide application in agriculture and public health. Its primary mode of causing harm is by irreversibly inhibiting acetylcholinesterase (AChE), an enzyme essential for the smooth running of the nervous system (Sakinah et al., 2024; Coppola et al., 2025). In addition to affecting AChE, CPF has also been linked to inducing oxidative stress and endocrine disruption, further expanding its spectrum of molecular toxicity. These secondary effects can further contribute to the toxicity and multisystem harm caused by exposure to this insecticide (Rathod and Garg, 2017).\u003c/p\u003e\n\u003cp\u003eOxidative stress is a major cause of many health problems, including diseases that affect the brain and nervous system, heart and blood vessels, and other parts of the body. It\u0026apos;s also linked to diabetes, cancer, and problems that come with getting older (Liguori et al., 2018). Moreover, exposure to environmental pollutants and xenobiotics such as pesticides like CPF (Uchendu et al., 2015) and BPA (Amjad et al., 2020) has been shown to induce oxidative stress.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Hesperidin (HES) is a potent bioflavonoid and naturally occurring antioxidant. Flavonoids are small molecular-weight compounds with phenolic structures found in medicinal plants and food items. They are abundant in nature and serve as one of the predominant bioactive constituents in citrus fruits (Noshy et al., 2022). Beyond its antioxidant capacity, HES also has a range of other benefits, including reducing inflammation, lowering blood pressure, fighting cancer, and protecting against cell damage (Pyrzynska, 2022; Osama et al., 2024), thus positioning it as a promising multifunctional bioactive compound with broad therapeutic potential.\u003c/p\u003e\n\u003cp\u003eThe extensive use of pesticides and the increasing consumption of BPA found in plastics pose new challenges regarding toxicity (hepato- and nephrotoxicity) and environmental pollution. Real-world environmental exposures to both humans and animals rarely occur in isolation; rather, they typically involve simultaneous or sequential exposure to multiple chemical agents, raising critical concerns about cumulative and potentially synergistic toxic effects of such co-exposures. Despite this reality, the overwhelming majority of toxicological research and safety assessments have historically focused on single chemicals in isolation. While such studies yield valuable data for individual chemical risk assessment, they fall critically short in capturing the complex toxicodynamic interactions and cumulative health burdens arising from co-exposure to chemicals in humans and animals (Bloch et al., 2023). \u0026nbsp;People are exposed to a wide range of chemicals every day through food, drinks, cosmetics, and indoor and outdoor pollutants. Studying the combined effects of low doses of multiple chemical contaminants on humans and animals is essential due to their widespread presence in the environment. It is also important to identify substances that may help reduce the negative health effects on the liver and kidneys following chronic co-exposure to low doses of these pollutants. As far as we know, this is the first time these two contaminants are being studied in combination.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty male Wistar rats, eight weeks old and weighing between 150 and 180 g, were sourced and housed in the Animal Holding Facility of the Veterinary Teaching Hospital, University of Jos, Jos, Nigeria. Prior to the commencement of the experiment, the animals were allowed a minimum acclimatization period of two weeks under standard laboratory conditions. Throughout the study, the rats were maintained on standard rat chow with unlimited access to clean drinking water. The experiment was done by following the rules set by the Institutional Animal Care and Use Committee of Animal Experimental Unit, Department of Pharmacology, University of Jos (UJ/FPS/F17-00379), as well as the guidelines for Care for Laboratory Animals (NRC 1996). 1996).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical Source \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChlorpyrifos, which is 20% emulsifiable concentration, sold as Termiphos\u0026reg; (Sabero organic, India) and corn oil (Wesson Corn oil\u0026reg;, U.S.A) were procured from reputable stores in Nigeria, while bisphenol A and hesperidin purchased from Sigma Aldrich, U.S.A. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental design \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty rats were randomly divided into six equal groups of five animals each. Group I (C/oil) was administered corn oil at 2 mlkg\u003csup\u003e-1\u003c/sup\u003e, while group II (CPF) was administered chlorpyrifos (CPF) at 4.75mgkg\u003csup\u003e-1\u003c/sup\u003e (corresponding to 1/20\u003csup\u003eth\u003c/sup\u003e LD\u003csub\u003e50\u003c/sub\u003e previously established by Uchendu et al. (2014). Group III (BPA) was administered Bisphenol A at 50 mgkg\u003csup\u003e-1\u003c/sup\u003eday\u003csup\u003e-1\u003c/sup\u003e (LOAEL for BPA in mammalian studies [Vandenberg et al., 2013]), while group IV (BPA +CPF) was co-administered BPA at 50mgkg\u003csup\u003e-1\u003c/sup\u003eday\u003csup\u003e-1\u003c/sup\u003e and CPF at 4.75mgkg\u003csup\u003e-1\u003c/sup\u003e. Group V (HES) was administered Hesperidin only at 100 mgkg\u003csup\u003e-1\u003c/sup\u003e, while group VI (HES+CPF+BPA) was pretreated with hesperidin at 100mgkg\u003csup\u003e-1\u003c/sup\u003e and then co- exposed to CPF at 4.75mgkg\u003csup\u003e-1\u003c/sup\u003e and BPA at 50mgkg\u003csup\u003e-1\u003c/sup\u003eday\u003csup\u003e-1\u003c/sup\u003e. All treatments were administered via oral gavage once daily for a duration of 16 weeks. \u003c/p\u003e\n\u003cp\u003eAt the end of the 16-week treatment period, the animals were humanely euthanized following light anaesthesia using ketamine at 50mgkg\u003csup\u003e-1\u003c/sup\u003e, and the blood sample was collected from each rat by jugular venesection. A small amount of blood, about 3 mL of blood was taken from each animal into a test tube. The blood was then left to clot at room temperature. After that, it was spun around at 800 x g in a centrifuge for 10 minutes. The serum samples was carefully taken out and put into new, clean test tubes. These tubes were then stored at -4\u003csup\u003eo\u003c/sup\u003eC until they were ready to be analyzed for any biochemical changes.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eSerum biochemical assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe serum samples were used to determine the concentrations of total protein, albumin, urea, and creatinine; aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities using a serum biochemistry autoanalyzer, Selectra XL (Netherlands). Globulin was derived by subtracting the values of albumin from total protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC-reactive protein concentration determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC-reactive protein (CRP) is a liver-derived acute-phase reactant that is released into circulation after tissue injury, infection or inflammation. The concentration of CRP was assayed in the serum samples using rat CRP ELISA kit (Abcam, UK) according to the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of oxidative stress biomarkers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe activities of serum antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)) were assessed using commercially available kits from Northwest Life Science Specialties Vancouver, LLC, Canada. \u003c/p\u003e\n\u003cp\u003eSerum SOD activity was measured using the method described by Martin et al. (1987). To assess SOD activity, we monitored the autoxidation rate of haematoxylin. By comparing the autoxidation rates in the presence and absence of the serum, we were able to determine SOD activity. The absorbance was measured at 560 nm every 10 minutes. The results were expressed as McCord-Fridovich \u0026apos;cytochrome c\u0026apos; units.\u003c/p\u003e\n\u003cp\u003eSerum CAT activity was determined according to the method of Beers and Sizer (1952) and expressed as IUL\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe activity of GPx in the serum was evaluated based on the method adapted from Paglia and Valentine (1967). In this method, GPx catalyzes the oxidation of glutathione by cumene hydroperoxide. The reduction in absorbance of nicotinamide adenine dinucleotide phosphate (NADPH) was measured at 340 nm in the presence of glutathione reductase and NADPH.\u003c/p\u003e\n\u003cp\u003eTo measure the amount of lipid peroxidation, MDA in the serum, the double heating method developed by Draper and Hadley (1990) and later modified by Yavuz et al. (2004) was used. This technique relies on spectrophotometric quantification of the chromogen formed by the reaction between thiobarbituric acid (TBA) and MDA. Absorbance was recorded at 532 nm with an ultraviolet spectrophotometer. The MDA concentration was calculated using the absorbance coefficient for the MDA-TBA complex, 1.56\u0026times;10\u003csup\u003e5\u003c/sup\u003e CM\u003csup\u003e-1\u003c/sup\u003eM\u003csup\u003e-1\u003c/sup\u003e and reported as nanomoles per milliliter. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKidney and liver samples were harvested at necropsy and fixed in 10% buffered formalin for 48 h. The tissues were subsequently processed and embedded in paraffin blocks using tissue processing procedures, as described by Luna (1968). Very thin slices about 4 \u0026mu;m thick were taken from each block and stained with hematoxylin and eosin (H\u0026amp;E). The stained sections were then looked at under a light microscope (Leica DM 1000, Germany) under x400 magnification. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are presented as mean \u0026plusmn; standard error of the mean (SEM). To see if there are any differences between groups, one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post-hoc test applied for multiple comparisons were used. All the data were analyzed using GraphPad Prism, version 5.0 for Windows (GraphPad Software, San Diego, California, USA). A probability value of P \u0026lt; 0.05 was considered significant. Sometimes, we also looked at percentages to show how things changed, when it was helpful to do so.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExperimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty male Wistar rats, eight weeks old and weighing between 150 and 180 g, were sourced and housed in the Animal Holding Facility of the Veterinary Teaching Hospital, University of Jos, Jos, Nigeria. Prior to the commencement of the experiment, the animals were allowed a minimum acclimatization period of two weeks under standard laboratory conditions. Throughout the study, the rats were maintained on standard rat chow with unlimited access to clean drinking water. The experiment was done by following the rules set by the Institutional Animal Care and Use Committee of Animal Experimental Unit, Department of Pharmacology, University of Jos (UJ/FPS/F17-00379), as well as the guidelines for Care for Laboratory Animals (NRC 1996). 1996).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical Source\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChlorpyrifos, which is 20% emulsifiable concentration, sold as Termiphos\u0026reg; (Sabero organic, India) and corn oil (Wesson Corn oil\u0026reg;, U.S.A) were procured from reputable stores in Nigeria, while bisphenol A and hesperidin purchased from Sigma Aldrich, U.S.A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental design\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty rats were randomly divided into six equal groups of five animals each. Group I (C/oil) was administered corn oil at 2 mlkg\u003csup\u003e-1\u003c/sup\u003e, while group II (CPF) was administered chlorpyrifos (CPF) at 4.75mgkg\u003csup\u003e-1\u003c/sup\u003e (corresponding to 1/20\u003csup\u003eth\u003c/sup\u003e LD\u003csub\u003e50\u003c/sub\u003e previously established by Uchendu et al. (2014). Group III (BPA) was administered Bisphenol A at 50 mgkg\u003csup\u003e-1\u003c/sup\u003eday\u003csup\u003e-1\u003c/sup\u003e (LOAEL for BPA in mammalian studies [Vandenberg et al., 2013]), while group IV (BPA +CPF) was co-administered BPA at 50mgkg\u003csup\u003e-1\u003c/sup\u003eday\u003csup\u003e-1\u003c/sup\u003e and CPF at 4.75mgkg\u003csup\u003e-1\u003c/sup\u003e. Group V (HES) was administered Hesperidin only at 100 mgkg\u003csup\u003e-1\u003c/sup\u003e, while group VI (HES+CPF+BPA) was pretreated with hesperidin at 100mgkg\u003csup\u003e-1\u003c/sup\u003e and then co- exposed to CPF at 4.75mgkg\u003csup\u003e-1\u003c/sup\u003e and BPA at 50mgkg\u003csup\u003e-1\u003c/sup\u003eday\u003csup\u003e-1\u003c/sup\u003e. All treatments were administered via oral gavage once daily for a duration of 16 weeks.\u003c/p\u003e\n\u003cp\u003eAt the end of the 16-week treatment period, the animals were humanely euthanized following light anaesthesia using ketamine at 50mgkg\u003csup\u003e-1\u003c/sup\u003e, and the blood sample was collected from each rat by jugular venesection. A small amount of blood, about 3 mL of blood was taken from each animal into a test tube. The blood was then left to clot at room temperature. After that, it was spun around at 800 x g in a centrifuge for 10 minutes. The serum samples was carefully taken out and put into new, clean test tubes. These tubes were then stored at -4\u003csup\u003eo\u003c/sup\u003eC until they were ready to be analyzed for any biochemical changes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerum biochemical assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe serum samples were used to determine the concentrations of total protein, albumin, urea, and creatinine; aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities using a serum biochemistry autoanalyzer, Selectra XL (Netherlands). Globulin was derived by subtracting the values of albumin from total protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC-reactive protein concentration determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC-reactive protein (CRP) is a liver-derived acute-phase reactant that is released into circulation after tissue injury, infection or inflammation. The concentration of CRP was assayed in the serum samples using rat CRP ELISA kit (Abcam, UK) according to the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of oxidative stress biomarkers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe activities of serum antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)) were assessed using commercially available kits from Northwest Life Science Specialties Vancouver, LLC, Canada.\u003c/p\u003e\n\u003cp\u003eSerum SOD activity was measured using the method described by Martin et al. (1987). To assess SOD activity, we monitored the autoxidation rate of haematoxylin. By comparing the autoxidation rates in the presence and absence of the serum, we were able to determine SOD activity. The absorbance was measured at 560 nm every 10 minutes. The results were expressed as McCord-Fridovich \u0026apos;cytochrome c\u0026apos; units.\u003c/p\u003e\n\u003cp\u003eSerum CAT activity was determined according to the method of Beers and Sizer (1952) and expressed as IUL\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe activity of GPx in the serum was evaluated based on the method adapted from Paglia and Valentine (1967). In this method, GPx catalyzes the oxidation of glutathione by cumene hydroperoxide. The reduction in absorbance of nicotinamide adenine dinucleotide phosphate (NADPH) was measured at 340 nm in the presence of glutathione reductase and NADPH.\u003c/p\u003e\n\u003cp\u003eTo measure the amount of lipid peroxidation, MDA in the serum, the double heating method developed by Draper and Hadley (1990) and later modified by Yavuz et al. (2004) was used. This technique relies on spectrophotometric quantification of the chromogen formed by the reaction between thiobarbituric acid (TBA) and MDA. Absorbance was recorded at 532 nm with an ultraviolet spectrophotometer. The MDA concentration was calculated using the absorbance coefficient for the MDA-TBA complex, 1.56\u0026times;10\u003csup\u003e5\u003c/sup\u003e CM\u003csup\u003e-1\u003c/sup\u003eM\u003csup\u003e-1\u003c/sup\u003e and reported as nanomoles per milliliter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKidney and liver samples were harvested at necropsy and fixed in 10% buffered formalin for 48 h. The tissues were subsequently processed and embedded in paraffin blocks using tissue processing procedures, as described by Luna (1968). Very thin slices about 4 \u0026mu;m thick were taken from each block and stained with hematoxylin and eosin (H\u0026amp;E). The stained sections were then looked at under a light microscope (Leica DM 1000, Germany) under x400 magnification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are presented as mean \u0026plusmn; standard error of the mean (SEM). To see if there are any differences between groups, one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s post-hoc test applied for multiple comparisons were used. All the data were analyzed using GraphPad Prism, version 5.0 for Windows (GraphPad Software, San Diego, California, USA). A probability value of P \u0026lt; 0.05 was considered significant. Sometimes, we also looked at percentages to show how things changed, when it was helpful to do so.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of treatments on liver function enzymes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe AST activity in the CPF group was significantly higher (P \u0026lt; 0.05) than in the C/oil group. Significant (P \u0026lt; 0.05) elevation was also observed in the BPA and CPF+BPA groups when compared to C/oil group. The AST activity in the CPF + BPA group was significantly higher (P \u0026lt; 0.05) than in the HES group. Notably, there was a significant decrease (P \u0026lt; 0.05) in the CPF + BPA+HES group compared to the CPF + BPA group.\u003c/p\u003e\n\u003cp\u003eSimilarly, the ALT activity in the BPA group was significantly higher than in the C/oil group. The activity in the CPF + BPA group was also significantly higher (P \u0026lt; 0.05) than in the HES group. There was no significant difference (P \u0026gt; 0.05) in the activity of ALT between CPF+BPA and CPF+BPA+HES groups, however, there was a 19.6% decrease in the CPF + BPA + HES group compared to the CPF + BPA group.\u003c/p\u003e\n\u003cp\u003eThere were no significant differences in ALP activity between the treatment groups. Although there was no significant difference (P \u0026gt; 0.05) between CPF+BPA and CPF+BPA+HES groups, it decreased by 15.7% in the CPF + BPA + HES group compared to the CPF + BPA group (Fig 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of treatments on urea concentration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentration of urea was significantly higher (P \u0026lt; 0.05) in the CPF + BPA group compared to those of C/oil, HES, CPF and BPA groups, respectively. Notably, there was a significant decrease (P \u0026lt; 0.05) in the CPF + BPA+ HES group compared to the CPF + BPA group (Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of treatments on creatinine concentration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCreatinine concentration was significantly increased (P \u0026lt; 0.05) in the CPF + BPA group compared to the C/oil, HES, BPA and the CPF + BPA + HES groups, respectively. There was a significant decrease (P \u0026lt; 0.05) in the CPF + BPA + HES group compared to the CPF + BPA (Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of treatments on serum proteins\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum total protein concentration was significantly elevated (P \u0026lt; 0.05) in the C/oil group when compared to the CPF group. Similarly, a significant increase (P \u0026lt; 0.05) was also observed in the HES group compared to the CPF group. There was no significant difference (P \u0026gt; 0.05) in serum total protein concentration between CPF+BPA and CPF+BPA+HES groups.\u003c/p\u003e\n\u003cp\u003eThe serum albumin concentration was significantly increased (P \u0026lt; 0.05) in the HES group compared to the CPF and CPF + BPA groups, respectively. There was no significant difference (P \u0026gt; 0.05) between CPF+BPA and CPF+BPA+HES groups, however, there was a 10. 5% increase in the albumin concentration in the CPF + BPA + HES group compared to the CPF + BPA group.\u003c/p\u003e\n\u003cp\u003eThere was a significant increase (P \u0026lt; 0.05) in the concentration of serum globulin in the CPF +BPA group compared to the C/oil, BPA, HES and CPF + BPA + HES groups, respectively. Notably, there was a significant decrease (P \u0026lt; 0.05) in the group treated with CPF + BPA + HES compared to the group treated with CPF + BPA (Fig. 4).\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of treatments on C - reactive protein concentration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe serum C-reactive protein concentration was significantly lesser (P \u0026lt; 0.05) in the C/oil group compared to the CPF, BPA and CPF + BPA groups, respectively. Similarly, there was significant increase (P \u0026lt; 0.05) in the CPF compared to the HES and CPF + BPA + HES groups, respectively. The concentration of C- reactive protein also significantly increased (P \u0026lt; 0.05) in the BPA, CPF + BPA and CPF + BPA + HES groups compared to the HES group. Notably, a significant decrease (P \u0026lt;0.05) was observed in the CPF + BPA + HES group compared to the CPF + BPA group (Fig. 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of treatments on malondialdehyde concentration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of MDA was much higher (P \u0026lt; 0.05) in the CPF group compared to the other groups, like the ones that got C/oil, BPA, CPF + BPA, HES, and CPF + BPA + HES. The amount of MDA was significantly lower (P \u0026lt; 0.05) in the groups that got HES and CPF + BPA + HES compared to the group that got CPF + BPA (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of treatments on the activities of antioxidant enzymes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCatalase activity exhibited a significant decrease in the CPF, BPA, CPF + BPA and CPF + BPA + HES groups (P \u0026lt; 0.05) compared to the C/oil group. Similarly, the activity also decreased significantly (P \u0026lt; 0.05) in the CPF group compared to the BPA, HES and CPF + BPA + HES groups, respectively. It was also noted that activity in the BPA group was much lower (P \u0026lt; 0.05) than in the CPF + BPA and HES groups. A significant (P \u0026lt; 0.05) decrease was recorded in the CPF + BPA and CPF + BPA + HES groups compared to the HES group. Notably, the activity in the CPF + BPA + HES group increased significantly (P \u0026lt; 0.05) compared to the CPF + BPA group (Table 1).\u003c/p\u003e\n\u003cp\u003eThe GPx activity in the CPF group significantly decreased (P \u0026lt; 0.05) compared to that of the C/oil, HES, and the CPF + BPA + HES groups, respectively. Similarly, the GPx activity in the HES group was significantly higher (P \u0026lt; 0.05) compared to the CPF + BPA group. Although the activity did not reach statistical significance (P \u0026gt; 0.05) between CPF+BPA and CPF+BPA+HES groups, it increased by 61.8% in the CPF+BPA+HES group compared to the CPF+BPA group (Table 1).\u003c/p\u003e\n\u003cp\u003eThe SOD activity was significantly decreased in the CPF and CPF + BPA groups (P \u0026lt; 0.05) compared to the C/oil group. In addition, the activity was much lower (P \u0026lt; 0.05) in the CPF group than in the HES and CPF + BPA + HES groups. The SOD activity also rose significantly (P \u0026lt; 0.05) in the HES group compared to the BPA and CPF + BPA groups, respectively. Although there was no significant (P \u0026gt; 0.05) difference in the activity between CPF+BPA and CPF+BPA groups, it was elevated by 46.8% in the CPF+BPA+HES group compared to the CPF+BPA group (Table 1).\u003c/p\u003e\n\u003cp\u003eThe GPx activity in the CPF group significantly decreased (P \u0026lt; 0.05) compared to that of the C/oil, HES, and the CPF + BPA + HES groups, respectively. Similarly, the GPx activity in the HES group was significantly higher (P \u0026lt; 0.05) compared to the CPF + BPA group. Although the activity did not reach statistical significance (P \u0026gt; 0.05) between CPF+BPA and CPF+BPA+HES groups, it increased by 61.8% in the CPF+BPA+HES group compared to the CPF+BPA group (Table 1).\u003c/p\u003e\n\u003cp\u003eThe SOD activity was significantly decreased in the CPF and CPF + BPA groups (P \u0026lt; 0.05) compared to the C/oil group. Furthermore, the activity was significantly lower (P \u0026lt; 0.05) in the CPF group compared to the HES and CPF + BPA + HES groups, respectively. The SOD activity also increased significantly (P \u0026lt; 0.05) in the HES group compared to the BPA and CPF + BPA groups, respectively. Although there was no significant (P \u0026gt; 0.05) difference in the activity between CPF+BPA and CPF+BPA groups, it was elevated by 46.8% in the CPF+BPA+HES group compared to the CPF+BPA group (Table 1).\u003cv:shapetype id=\"_x0000_t202\" coordsize=\"21600,21600\" o:spt=\"202\" path=\"m,l,21600r21600,l21600,xe\"\u003e\n \u003cv:stroke joinstyle=\"miter\"\u003e\n \u003cv:path gradientshapeok=\"t\" o:connecttype=\"rect\"\u003e\u0026nbsp;\u003c/v:path\u003e\u0026nbsp;\n \u003c/v:stroke\u003e\u0026nbsp;\n \u003c/v:shapetype\u003e\n \u003cv:shape id=\"Text_x0020_Box_x0020_46\" o:spid=\"_x0000_s1027\" type=\"#_x0000_t202\" 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filled=\"f\" stroked=\"f\" strokeweight=\".5pt\"\u003e\u0026nbsp;\u003cv:textbox\u003e\u0026nbsp;\u003c/v:textbox\u003e\u0026nbsp;\u003c/v:shape\u003e\n\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological findings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe liver tissue of the corn oil (C/oil) and hesperidin (HES) groups showed normal histological appearance with hepatocellular architecture intact (Fig. 6. 1,5). Examination of liver tissue in rats given CPF showed mild loss of hepatocytes (L) and mild focal infiltration of inflammatory cells (IC) (Fig. 6. 2). Moderate congestion (C), mild loss of hepatocytes (L) and moderate accumulation of eosinophilic material in the hepatic vein (E) were observed in the group exposed to BPA only (Fig 6. 3). Examination of liver tissue of rats given CPF + BPA showed moderate perivascular infiltration of inflammatory cells (IC), moderate congestion (C) and mild necrosis of hepatocytes (N) (Fig.6. 4). Mild hepatocellular necrosis (N), moderate congestion (C) with mild infiltration of inflammatory cells (IC) were detected in the CPF+BPA+HES group (Fig. 6. 6).\u003c/p\u003e\n\u003cp\u003eThe kidney tissues of the corn oil and HES groups showed normal renal architecture (Fig. 7. 1, 5). Examination of the kidney tissues of rats given CPF only showed moderate fragmentation of the glomeruli (F) and mild congestion (C) (Fig. 7. 2). Moderate desquamation and loss of tubular epithelial cells (A) with mild focal infiltration of inflammatory cells (IC) were detected in the group exposed to BPA only (Fig. 7. 3). Examination of kidney tissue of rats co-exposed to CPF + BPA showed mild perivascular infiltration of inflammatory cells (IC) and moderate loss of tubular epithelial cells (A) (Fig. 7. 4). Mild loss of tubular epithelial cells (A) were observed in the group administered CPF+BPA+HES (Fig. 7. 6).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eExposure to environmental contaminants such as pesticides and BPA at low doses poses significant health risks worldwide, as their exposure has been well-documented to inflict damage across multiple organ systems in both humans and animals. Due to their pivotal roles in the metabolism and excretion of foreign substances, the liver and kidneys have traditionally been viewed as the primary targets for various chemicals that cause toxic effects after environmental exposure. The cells of these organs are exposed to significant concentrations of chemicals, and chemical-induced hepatotoxicity and nephrotoxicity have emerged as a growing concern of global public health significance. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe present study found that chronic exposure to CPF and/or BPA caused a significant increase in the activities of liver-associated enzymes, specifically AST, ALT, and ALP. Chronic co-exposure to CPF and BPA (CPF + BPA) showed the most significant elevation in AST, ALP, and ALT activity compared to exposure to either of the chemicals alone. The elevated liver enzyme activities could be linked to liver dysfunction and significant changes in the liver membrane, as evidenced by mild necrosis of the hepatocytes and moderate perivascular infiltration of inflammatory cells and congestion observed histopathologically. Previous research has reported elevated levels of liver-related enzyme activities in rats exposed to CPF alone (Zhang et al., 2021; Saoudi et al., 2021) and BPA alone (Eweda et al., 2020; Liu et al., 2022). Alanine aminotransferase is considered the most reliable and specific biomarker of hepatocellular injury, given its predominant localization within hepatocytes. Accordingly, the observed increase in ALT activity likely reflects underlying degenerative and cytotoxic changes in the liver parenchyma and associated tissues, indicative of compromised hepatocellular function. Increased serum ALP activity has been associated with pathological modifications not only in the liver but also in the bones, kidneys, intestines, and leucocytes, indicating potential multi-organ damage. The elevated ALP activity may result from oxidative damage affecting these indicated organs or direct interactions of the pesticide (Uchendu et al., 2015; Sule et al., 2022) and BPA (Liu et al., 2022). Aspartate aminotransferase is a crucial enzyme that helps turn aspartate and alpha-ketoglutarate into oxaloacetate and glutamate, playing an essential role in amino acid and energy metabolism. Accumulating evidence further indicates that BPA exposure induces hepatic injury through oxidative stress (Abdulhameed et al., 2022), with particular impact on mitochondrial integrity, alongside lipid peroxidation and inflammatory cascades. The increase observed in the activities of these enzymes may be due to oxidative damage to the liver parenchyma, as the liver is known to be centrally involved in the primary site of xenobiotic detoxification and is chronically exposed to xenobiotics and their reactive metabolites. Consequently, observed elevation in hepatic enzyme activities is likely attributable to impaired hepatocellular function and disruptions in enzyme biosynthesis, resulting in changes in the permeability of the hepatic membrane and the subsequent leakage of intracellular enzymes into the systemic circulation (Ashoush et al., 2020). Furthermore, co-exposure to CPF and BPA may have caused a release of these enzymes from the liver\u0026apos;s cytosol into the bloodstream as a result of the pathological lesions induced in the study. Supplementation with HES attenuated the elevated hepatic enzyme activities observed in the co-exposed group, attributed to the well-documented hepato-protective properties of HES against tissue injuries. This protective effect was further corroborated histopathologically by the markedly reduced incidence and severity of pathological lesions observed in the HES-treated group. Additionally, HES have been shown to mitigate cellular damage by enhancing cellular defense mechanisms (Nasehi et al., 2023). The hepato-protective effect of HES through its ability to reduce inflammation and oxidative stress has been documented by Tabeshpour et al. (2020).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The present study demonstrated that prolonged exposure to both CPF and BPA resulted in a significant elevation in serum urea concentrations. Urea is primarily filtered and excreted by the kidneys as a metabolic waste product. Therefore, the increased concentration of this metabolite after exposure to CPF and BPA may serve as a sensitive indicator of compromised renal function. Albasher et al. (2019), Afzal et al. (2022), and Sakinah et al. (2024) have shown that elevated urea concentration following CPF exposure may be a result of impaired glomerular filtration alongside tubular reabsorption. Studies have also shown that elevated urea concentration following BPA exposure may be attributed to impaired glomerular filtration and tubular function (Kobroob et al., 2018; Mohammed, 2023). The observed moderate fragmentation of the glomeruli, desquamation, and loss of tubular epithelial cells, as well as the mild congestion and focal infiltration of inflammatory cells in the rats exposed to the combination of CPF and BPA further confirm the elevated urea concentration in this study. The study demonstrates that supplementation with HES provided renal protection by significantly reducing the heightened level of urea observed in the co-exposed (CPF + BPA) group. This was also exemplified by the mild loss of tubular epithelial cells observed in the group. This finding is congruent with the works of Kucukler et al. (2021) who stated that HES reduced the high urea concentration in rats chronically exposed to CPF, and Hassan et al. (2023) who also demonstrated the protective effect of HES against aluminum-provoked renal injury in rats by reducing the increased urea concentration.\u003c/p\u003e\n\u003cp\u003eThe present study further demonstrated that continuous prolonged exposure to both CPF and BPA led to an increase in serum creatinine concentration. This rise in creatinine concentration provides compelling evidence of nephrotoxic injury within the renal parenchyma, as indicated by moderate fragmentation of the glomeruli, desquamation and loss of tubular epithelial cells, as well as mild congestion and focal infiltration of inflammatory cells. Previous research has also reported increased creatinine levels following exposure to CPF (Owumi and Dim, 2019; Sakinah et al., 2024) and BPA (Kobroob et al., 2018; Mohammed, 2023). Creatinine, an end-product of creatine phosphate catabolism in skeletal muscle under normal physiological conditions, is a reliable endogenous marker of glomerular filtration rate. The kidneys are responsible for removing creatinine from the body, with minimal reabsorption in the tubules. If the glomerular filtration rate decreases due to renal impairment, creatinine tends to accumulate in the bloodstream (Braun et al., 2003; Albasher et al., 2019). The rise in serum creatinine levels was more significant in the group exposed to both CPF and BPA compared to when either CPF or BPA was administered alone, suggesting that the combination caused more damage to the kidneys and/or muscles. Pretreatment with HES helped reduce the elevated creatinine concentration in the CPF + BPA group. This may be due to the anti-inflammatory and antioxidant properties of HES against CPF and BPA-induced renal and muscle damage, as evidenced by the mild loss of tubular epithelial cells observed histopathologically in this group. Hesperidin has been reported to alleviate renal damage by reducing vascular lesions, tubular cell vacuolation, and tissue damage caused by chemical contaminants (Hassan et al., 2023). Osama et al. (2024) also reported a decrease in creatinine concentration following HES supplementation in nephrotoxicity provoked by carbimazole induced hypothyroidism in adult rats.\u003c/p\u003e\n\u003cp\u003eThe present study demonstrated that the CPF exposure group exhibited significantly reduced total protein and albumin concentrations compared to both the co-exposed (CPF+BPA) and control group. This decrease in serum protein and albumin levels is likely due to CPF and BPA-induced hepatocellular damage, as evidenced by increased hepatic enzyme activities, mild hepatocyte necrosis, moderate congestion, and infiltration of inflammatory cells observed in the study. The observed low albumin and protein levels are consistent with previous research after exposure to CPF and Deltamethrin pesticides (Uchendu et al., 2015) and BPA (Geetharath and Josthna, 2016). Impaired hepatic albumin biosynthesis secondary to liver dysfunction or enhanced renal albumin loss through compromised tubular reabsorption, as suggested by Uchendu et al. (2015) may be responsible. Furthermore, the impact on protein integrity is more apparent than its synthesis due to BPA\u0026apos;s induction of mitochondrial oxidative stress, leading to proteolytic degradation of existing protein pools. This is exacerbated by BPA disrupting hepatic integrity and function, collectively culminating in marked reductions (Abdel-Rahman et al., 2018). Supplementation with HES helped alleviate the decrease in serum total protein and albumin caused by CPF+BPA. This may be due to HES\u0026apos;s antioxidative abilities, including\u0026nbsp;its ability to fight off harmful free radicals and boost the body\u0026apos;s own antioxidant defenses. Studies have shown that HES neutralizes various ROS, protecting proteins from oxidative damage (Hajialyani et al., 2019). Hesperidin has also demonstrated renoprotective attributes against oxidative damage (Khan and Parvez, 2015) by potentially reducing protein loss through renal pathways.\u003c/p\u003e\n\u003cp\u003eC-reactive protein (CRP) is a pentraxin-family acute phase reactant that rises rapidly in response to tissue injury, infection, and systemic inflammation (Wu et al., 2016). Acute phase proteins, including CRP, are widely known for their diagnosis, monitoring, and prognostic assessment of a broad spectrum of acute and chronic disease conditions (Yaqub et al., 2023). The significant elevation in circulating CRP levels indicates sustained systemic inflammation and ongoing tissue injury induced by these xenobiotics. C-reactive protein can be used alongside other markers of inflammation for comprehensive assessment of both acute and chronic inflammatory burden (Yaqub et al., 2023). The CRP level was significantly increased in the CPF+BPA group compared to the individual treatment groups. Elevated C-reactive protein concentrations have been reported following CPF (Yaqub et al., 2023) and BPA (Tsen et al., 2021) exposure. The elevated concentration may be a response to specific inflammatory cytokines such as interleukin-6, likely associated with hepatocellular damage and oxidative stress induced by these contaminants in the present study. Supplementation with HES resulted in a significant drop in CRP levels. This effect may be explained by the inflammation reducing and antioxidant properties of HES. Previous studies have demonstrated that HES inhibits inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, leading to decreased prostaglandin levels and reduced inflammation. These multi-faceted mechanisms of HES likely contribute to the observed reduction in CRP concentration in the co-exposed group (Khorasanian et al., 2023).\u003c/p\u003e\n\u003cp\u003eThe present study showed that exposure to CPF and/or BPA increased the levels of MDA in the serum of rats. The CPF-exposed group had significantly higher MDA levels compared to the group exposed to both CPF and BPA. The observed increase in serum MDA concentration is likely due to elevated reactive oxygen species (ROS) levels and the inhibition of serum antioxidant enzyme activities identified in this study. Impairment of the enzymatic antioxidant system results in the accumulation of free radicals, which promote increased lipid peroxidation (LPO) upon exposure to environmental contaminants. Malondialdehyde, a principal product of polyunsaturated fatty acid (PUFA) peroxidation, serves as a key indicator of LPO induced by ROS in the body (Akpa et al., 2021). Chlorpyrifos and BPA, both lipophilic substances, may further enhance LPO through direct interactions with cellular membranes. The observed elevation in serum MDA concentration aligns with findings reported by other researchers following chlorpyrifos (CPF) (Kaur and Jindal, 2017) and BPA (Eweda et al., 2020) exposures. Elevated MDA concentrations following CPF exposure indicate an increase in LPO, which could potentially cause significant damage to cells and compromise the integrity of their cell membranes (Akpa et al., 2021). These reactions are closely linked to physiological functions and can act as both triggers and outcomes of cellular damage, with a well-established connection between oxidative stress and inflammation. Supplementation with HES was effective in reducing MDA levels in the CPF+BPA group. This result can be attributed to the antioxidant properties of HES, which help scavenge free radicals and increase antioxidant activities in the serum to detoxify free radicals. These findings align with those of Naseh et al. (2023) and Homayouni et al. (2017), who reported the effectiveness of HES against liver fibrosis induced by bile duct ligation in rats and Type 2 Diabetes, respectively.\u003c/p\u003e\n\u003cp\u003eAlthough the body possesses complex inherent antioxidant defense mechanisms, exposure to xenobiotics often leads to an overproduction of reactive oxygen species (ROS) in both the intracellular and extracellular environments. This overproduction can surpass the body\u0026apos;s natural ability to counteract oxidative damage. The present study revealed altered antioxidant enzyme activities (CAT, GPx, and SOD) in the serum of rats chronically exposed to CPF and/or BPA. The decline in SOD activity might be attributed to the direct effect of CPF and BPA or as a result of the production of free radicals induced by the contaminants. Superoxide dismutase promotes the breakdown of superoxide radicals, which are generated as consequence of heightened metabolic activity triggered by xenobiotics (Albasher et al., 2019). Superoxide dismutase also facilitates the breakdown of superoxide radicals into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e. It serves as the initial enzyme involved in managing oxidative radicals (Kaur, 2017). Several authors have previously reported a reduction in SOD activity in rats exposed to CPF (Kopjar et al., 2018) and BPA (Olujimi et al., 2020). The enhanced consumption of SOD during CPF+BPA-induced autoxidation could contribute to the decline in serum SOD activity. Such reduction in SOD activity might lead to an accumulation of superoxide radicals, potentially deactivating GPx and escalating hydrogen peroxide production (Abdel-Rahman et al., 2018; Meli et al., 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe present study also demonstrated that exposure to CPF and/or BPA resulted in inhibition of the activity of GPx. Glutathione peroxidase is an antioxidant enzyme that contains selenium whose biological role is to protect against oxidative damage via its aiding of the conversion of hydrogen peroxide into water. The decrement in GPx activity observed in this study could potentially arise from oxidative inactivation caused by the accumulation of CPF+BPA within the body. This oxidative stress could stem from GPx inhibition, often followed by decreased levels of GSH or an elevation in hydrogen peroxide production. Several authors have previously reported a reduction in GPx activity in rats exposed to CPF (Akpa et al., 2021) and BPA (Eweda et al., 2020). Reduced GPx activity induced by CPF and BPA correlates with elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels and the direct suppression of SOD function. This connection underscores the impact of diminished GPx activity in raising H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels within the body and concurrently inhibiting SOD\u0026apos;s effectiveness. Taken together, these findings reveal the intricate interrelation between GPx, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and SOD in regulating oxidative processes (Eweda et al., 2020).\u003c/p\u003e\n\u003cp\u003eThe decline in CAT activity in this study is likely connected to the decrease induced by CPF and BPA in the activities of SOD since SOD is responsible for converting superoxide anions into hydrogen peroxide (Akpa et al., 2021). The decline in catalase activity could also result from the enzyme\u0026apos;s depletion while trying to neutralize the hydrogen peroxide produced following co-exposure to CPF and BPA. Another factor could be the deactivation of the enzyme due to the excessive production of ROS in mitochondria and microsomes (Aboul Ezz et al., 2015). Several authors have previously reported a reduction in CAT activity in rats exposed to CPF (Albasher et al., 2019) and BPA (Aboul Ezz et al., 2015). However, the alleviation of the changes in serum antioxidant enzyme activities (SOD, GPx, and CAT) provoked by CPF+DLT exposure following HES supplementation may be due to the antioxidant attributes of HES. Hesperidin can scavenge and eliminate free radicals, effectively curtailing the buildup of free radical, and bolstering the body\u0026apos;s inherent antioxidant defenses, possibly by amplifying the activities of endogenous antioxidant enzymes such as SOD, CAT, and GPx. This dual approach serves to protect cells from oxidative stress and potential injury (Estruel-Amades et al., 2019). Prior studies by the following authors (Pari et al. 2014; Khan and Parvez 2015; Celik et al. 2016; Caglayan et al. 2019; Nasehi et al., 2023) have consistently reported increased SOD, GPx, and CAT activities following HES supplementation in various toxicity-induced models, highlighting HES potential in counteracting oxidative stress and bolstering antioxidant defenses.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In conclusion, exposure of rats to low doses of both CPF and BPA over a period of time, caused more alterations in certain serum biochemical parameters (such as AST, ALP, ALT activities, and urea, creatinine, and CRP levels) compared to those exposed to just one of the contaminants. It can be inferred that the co-exposure to CPF and BPA resulted in more negative effects by triggering inflammation and oxidative stress. The use of HES as a supplement helped improve the impaired kidney and liver functions, partly by reducing urea, creatinine, and CRP levels, as well as AST, ALT, and ALP activities. Additionally, it helped mitigate oxidative stress by decreasing LPO, thereby restoring the rats\u0026apos; antioxidant status.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALP Alkaline phosphatase\u003c/p\u003e\n\u003cp\u003eALT Alanine aminotransferase\u003c/p\u003e\n\u003cp\u003eANOVA One-way analysis of variance \u003c/p\u003e\n\u003cp\u003eAST Aspartate aminotransferase\u003c/p\u003e\n\u003cp\u003eBPA Bisphenol A\u003c/p\u003e\n\u003cp\u003eCAT Catalase\u003c/p\u003e\n\u003cp\u003eCPF Chlorpyrifos\u003c/p\u003e\n\u003cp\u003eCRP C-reactive proteins\u003c/p\u003e\n\u003cp\u003eDLT Deltamethrin\u003c/p\u003e\n\u003cp\u003eGPx Glutathione peroxidase\u003c/p\u003e\n\u003cp\u003eHE Hematoxylin eosin\u003c/p\u003e\n\u003cp\u003eHES Hesperidin\u003c/p\u003e\n\u003cp\u003eIL-1 Interleukin-1\u003c/p\u003e\n\u003cp\u003eiNOS inducible nitric oxide synthase\u003c/p\u003e\n\u003cp\u003eLD50 Median lethal dose 50 \u003c/p\u003e\n\u003cp\u003eLOAEL Lowest observed adverse effect level\u003c/p\u003e\n\u003cp\u003eLPO Lipid peroxidation\u003c/p\u003e\n\u003cp\u003eMDA Malondialdehyde \u003c/p\u003e\n\u003cp\u003eROS Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eSEM Standard error of the mean\u003c/p\u003e\n\u003cp\u003eSOD Superoxide dismutase\u003c/p\u003e\n\u003cp\u003eTBA Thiobarbituric acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements: \u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was sort for use of animals from the Animal Care and Use Committee of Animal Experimental Unit of the Department of Pharmacology, University of Jos, Nigeria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent of publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and materials are available on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e Funding sources \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no specific funding for this work. \u003c/p\u003e\n\n\n\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"683\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 183px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNames of Authors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 500px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eContributions of Authors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 183px;\"\u003e\n \u003cp\u003eEnokela Shaibu IDOGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 500px;\"\u003e\n \u003cp\u003eImplementation of the research.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 183px;\"\u003e\n \u003cp\u003eNendirmwa Musa DASHE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 500px;\"\u003e\n \u003cp\u003eDesign of the work, implementation of the research and drafting of manuscript.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 183px;\"\u003e\n \u003cp\u003eOnuche Shalom AGWECHE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 500px;\"\u003e\n \u003cp\u003eImplementation of the research.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 183px;\"\u003e\n \u003cp\u003eBlessing EDOGBO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 500px;\"\u003e\n \u003cp\u003eDrafting of manuscript and review.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 183px;\"\u003e\n \u003cp\u003eJoy Iyojo ITODO.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 500px;\"\u003e\n \u003cp\u003eDrafting of manuscript and review.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 183px;\"\u003e\n \u003cp\u003eChidiebere UCHENDU \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 500px;\"\u003e\n \u003cp\u003eDesign of the work, implementation of the research, data analysis and interpretation, writing of manuscript and review. \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdel-Rahman, H.G., Abdelrazek, H.M.A., Zeidan, D.W., Mohamed, R.M. \u0026amp; Abdelazim, A.M. (2018). Lycopene: hepatoprotective and antioxidant effects toward bisphenol A-induced toxicity in female Wistar rats\u003cem\u003e.\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003eOxidative Medicine and Cellular Longevity,\u003c/em\u003e doi: 10.1155/2018/5167524. \u003c/li\u003e\n\u003cli\u003eAbdulhameed, A.A.R., Lim, V., Bahari, H., Khoo, B.Y., Abdullah, M.N.H., Tan, J.J. \u0026amp;Yong, Y.K. (2022). Adverse effects of bisphenol A on the liver and its underlying mechanisms: evidence from \u003cem\u003ein-vivo\u003c/em\u003e and \u003cem\u003ein-vitro\u003c/em\u003e studies\u003cem\u003e.\u003c/em\u003e \u003cem\u003eBiomed Research International, doi: 10.1155/2022/8227314.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eAboul Ezz, H.S., Khadrawy, Y.A. \u0026amp; Mourad, I.M. (2015). The effect of bisphenol A on some oxidative stress parameters and acetylcholinesterase activity in the heart of male albino rats. \u003cem\u003eCytotechnology,\u003c/em\u003e 67,145-155. doi: 10.1007/s10616-013-9672-1.\u003c/li\u003e\n\u003cli\u003eAdiga, D., Nadeem Khan, G., Eswaran, S., Sriharikrishnaa, S., Chakrabarty, S., Rai, P.S. \u0026amp; Kabekkodu, S.P. (2022). Bisphenol A associated signaling pathways in human diseases. In: Gassman NR (ed) Bisphenol A: a multi-modal endocrine disruptor. Royal Society of Chemistry Cambridge, pp 42\u0026ndash;86. https://doi.org/10.1039/9781839166495-00042.\u003c/li\u003e\n\u003cli\u003eAkpa, A.R., Ayo, J.O., Mika\u0026rsquo;il, H.G. \u0026amp; Zakari, F.O. (2021). Protective effect of fisetin against subchronic chlorpyrifos-induced toxicity on oxidative stress biomarkers and neurobehavioral parameters in adult male albino mice. \u003cem\u003eToxicological Research,\u003c/em\u003e 37, 163-171. doi.org/10.1007/s43188-020-00049-y.\u003c/li\u003e\n\u003cli\u003eAlbasher, G., Almeer, R., Alarifi, S., Alkhtani, S., Farhood, M., Al-Otibi, F.O. \u0026amp; Rizwana, H. (2019). Nephroprotective role of \u003cem\u003eBeta vulgaris L\u003c/em\u003e. root extract against chlorpyrifos-induced renal injury in rats. \u003cem\u003eEvidence Based Complementary and Alternative Medicine,\u003c/em\u003e doi: 10.1155/2019/3595761. \u003c/li\u003e\n\u003cli\u003eAmjad, S., Rahman, M.S. \u0026amp; Pang, M.G. (2020). Role of antioxidants in alleviating bisphenol A toxicity. \u003cem\u003eBiomolecules,\u003c/em\u003e 10(8), 1105. doi: 10.3390/biom10081105.\u003c/li\u003e\n\u003cli\u003eAshoush, Y., Abozid, M., Mansour, S. \u0026amp; Morgan, A. (2020). Effect of chlorpyrifos on liver function of albino rates. \u003cem\u003eMenoufia Journal of Agricultural Biotechnology,\u003c/em\u003e 5(2), 83-92. doi: 10.21608/MJAB.2020.170415.\u003c/li\u003e\n\u003cli\u003eBala, R., Dhingra, S., Kumar, M., Bansal, K., Mittal, S., Sharma, R.K. \u0026amp; Wangoo, N. (2017). Detection of organophosphorus pesticide\u0026ndash;Malathion in environmental samples using peptide and aptamer based nanoprobes. \u003cem\u003eChemical Engineering Journal,\u003c/em\u003e 311, 111-116. doi.10.1016/j.cej.2016.11.070.\u003c/li\u003e\n\u003cli\u003eBeers, R.F. \u0026amp; Sizer, I.W. (1952). A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. \u003cem\u003eJournal of Biological Chemistry,\u003c/em\u003e 195(1), 133-140.\u003c/li\u003e\n\u003cli\u003eBloch, D., Diel, P., Epe, B., Hellwig, M., Lampen, A., Mally, A., Marko, D., Villar Fern\u0026aacute;ndez, M.A., Guth, S., Roth, A., Marchan, R., Ghallab, A., Cadenas, C., Nell, P., Vartak, N., van Thriel, C., Luch, A., Schmeisser, S., Herzler, M., Landsiedel, R., Leist, M., Marx‑Stoelting, P., Tralau, T. \u0026amp; Hengstler, J.G. (2023). Basic concepts of mixture toxicity and relevance for risk evaluation and regulation. \u003cem\u003eArchives of Toxicology, \u003c/em\u003e97, 3005\u0026ndash;3017. https://doi.org/10.1007/s00204-023-03565-6.\u003c/li\u003e\n\u003cli\u003eBraun, J.P., Lefebvre, H.P. \u0026amp; Watson, A.D.J. (2003). Creatinine in the dog: a review. \u003cem\u003eVeterinary Clinical Pathology,\u003c/em\u003e 32(4), 162-179. doi: 10.1111/j.1939-165x.2003.tb00332.x.\u003c/li\u003e\n\u003cli\u003eCaglayan, C., Demir, Y., Kucukler, S., Taslimi, P., Kandemir, F.M. \u0026amp; Gul\u0026ccedil;in, İ. (2019). The effects of hesperidin on sodium arsenite‐induced different organ toxicity in rats on metabolic enzymes as antidiabetic and anticholinergics potentials: A biochemical approach. \u003cem\u003eJournal of Food Biochemistry,\u003c/em\u003e 43(2), e12720. doi: 10.1111/jfbc.12720.\u003c/li\u003e\n\u003cli\u003eCelik, E., Oguzturk, H., Sahin, N., Turtay, M.G., Oguz, F. \u0026amp; Ciftci, O. (2016). Protective effects of hesperidin in experimental testicular ischemia/reperfusion injury in rats. \u003cem\u003eArchives of Medical Science,\u003c/em\u003e 12(5), 928-934. doi: 10.5114/aoms.2015.47697.\u003c/li\u003e\n\u003cli\u003eChoi, C.W., Jeong, J.Y., Hwang, M.S., Jung, K.K., Lee, K.H. \u0026amp; Lee, H.M. (2010). Establishment of the Korean tolerable daily intake of bisphenol A based on risk assessments by an expert committee. \u003cem\u003eToxicological Research,\u003c/em\u003e 26(4), 285-291. doi.org/10.5487/TR.2010.26.4.285.\u003c/li\u003e\n\u003cli\u003eCoppola, L., Lori, G., Talt, S., Sogorb, M.A. \u0026amp; Estevan, C. (2025). Evaluation of developmental toxicity of chlorpyrifos through new approach methodologies: a systematic review. \u003cem\u003eArchives of Toxicology\u003c/em\u003e, 99, 935\u0026ndash;981. doi.org/10.1007/s00204-024-03945-6.\u003c/li\u003e\n\u003cli\u003eCosta, H.E., Medeiros, I., Mariana, M. \u0026amp; Cairrao. E. (2025). Maternal\u0026ndash;foetal effects of exposure to Bisphenol A: outcomes and long-term consequence. \u003cem\u003eApplied Sciences\u003c/em\u003e, 15,697. https://doi.org/10.3390/ app15020697.\u003c/li\u003e\n\u003cli\u003edas Neves, R.N., Carvalho, F., Carvalho, M., Fernandes, E., Soares, E., de Bastos, M.L. \u0026amp; de Pereira, M.L. (2004). Protective activity of hesperidin and lipoic acid against sodium arsenite acute toxicity in mice. \u003cem\u003eToxicologic Pathology,\u003c/em\u003e 32(5), 527-35. doi: 10.1080/01926230490502566. \u003c/li\u003e\n\u003cli\u003e\u003c/li\u003e\n\u003cli\u003eDraper, H.H. \u0026amp; Hadley, M. (1990). Malondialdehyde determination as index of lipid Peroxidation\u003cem\u003e. \u003c/em\u003eIn Methods in \u003cem\u003eEnzymology, \u003c/em\u003e186, 421-431.\u003c/li\u003e\n\u003cli\u003eEl-Gameel, D., Hamdy, N.A., El-Yazbi, A.F., Ghanem, M.A., El-Khordaugi, L.K., Abdallah, S.M., Mechref, Y. \u0026amp; El-Yazbi, A.F. (2024). Chronic exposure to organophosphate pesticides and elevated markers of systemic inflammation: Possible neuroinflammatory and genotoxic effects. \u003cem\u003eJournal of Pharmacology and Experimental Therapeutics,\u003c/em\u003e 385 (S3) 27. doi.org/10.1124/jpet.122.190690.\u003c/li\u003e\n\u003cli\u003eEstruel-Amades, S., Massot-Cladera, M., Garcia-Cerd\u0026agrave;, P., P\u0026eacute;rez-Cano, F.J., Franch, \u0026Agrave;. Castell, M. \u0026amp; Camps-Bossacoma, M. (2019). Protective effect of hesperidin on the oxidative stress induced by an exhausting exercise in intensively trained rats. \u003cem\u003eNutrients,\u003c/em\u003e 11(4), 783.doi: 10.3390/nu11040783.\u003c/li\u003e\n\u003cli\u003eEweda, S.M., Newairy, A.S.A., Abdou, H.M. \u0026amp; Gaber, A.S. (2020). Bisphenol A‑induced oxidative damage in the hepatic and cardiac tissues of rats: The modulatory role of sesame lignans. \u003cem\u003eExperimental and Therapeutic Medicine,\u003c/em\u003e 19(1), 33-44. doi: 10.3892/etm.2019.8193.\u003c/li\u003e\n\u003cli\u003eGeetharathan, T. \u0026amp; Josthna, P. (2016). Effect of BPA on protein, lipid profile and immuno-histo chemical changes in placenta and uterine tissues of albino rat\u003cem\u003e.\u003c/em\u003e \u003cem\u003eInternational Journal of Pharmaceutical and Clinical Research,\u003c/em\u003e 8(4), 260-268.\u003c/li\u003e\n\u003cli\u003eGrandjean, P. \u0026amp; Landrigan, P.J. (2014). Neurobehavioural effects of developmental toxicity. \u003cem\u003eThe\u003c/em\u003e \u003cem\u003eLancet Neurology,\u003c/em\u003e 13(3), 330-338. doi: 10.1016/S1474-4422(13)70278-3.\u003c/li\u003e\n\u003cli\u003eHajialyani, M., Hosein Farzaei, M., Echeverr\u0026iacute;a, J., Nabavi, S.M., Uriarte, E. \u0026amp; Sobarzo-S\u0026aacute;nchez, E. (2019). Hesperidin as a neuroprotective agent: a review of animal and clinical evidence. \u003cem\u003eMolecules,\u003c/em\u003e 24(3), 648. doi: 10.3390/molecules24030648.\u003c/li\u003e\n\u003cli\u003eHassan, Z.K., Elobeid, M.A., Virk, P., Omer, S.A., Elamin, M., Daghestani, M.H. \u0026amp; Alolayan, E.M. (2012). Bisphenol A induces hepatotoxicity through oxidative stress in rat model. \u003cem\u003eOxidative Medicine and Cellular Longevity,\u003c/em\u003e doi: 10.1155/2012/194829. \u003c/li\u003e\n\u003cli\u003eHassan, N.H., Yousef, D.M. \u0026amp; Alsemeh, A.E. (2023). Hesperidin protects against aluminum-induced renal injury in rats via modulating MMP-9 and apoptosis: biochemical, histological, and ultrastructural study\u003cem\u003e. Environmental Science and Pollution Research International,\u003c/em\u003e 30(13), 36208-36227. doi: 10.1007/s11356-022-24800-0.\u003c/li\u003e\n\u003cli\u003eHelal, E.G.E., Badawi, M.M.M., Soliman, M.G., Abdel-Kawi, N.A., Fadel, H.A.E. \u0026amp; Abozaid, N.M.G. (2013). Physiological and histopathological studies on bisphenol-A compound as xenoestrogen in male albino rats. \u003cem\u003eThe Egyptian Journal of Hospital Medicine,\u003c/em\u003e 50, 127 \u0026ndash; 136.doi. 10.21608/EJHM.2018.16081.\u003c/li\u003e\n\u003cli\u003eHomayouni, F., Haidari, F., Hedayati, M., Zakerkish, M. \u0026amp; Ahmadi, K. (2017). Hesperidin supplementation alleviates oxidative DNA damage and lipid peroxidation in type 2 diabetes: A randomized double‐blind placebo‐controlled clinical trial. \u003cem\u003ePhytotherapy Research,\u003c/em\u003e 31(10), 1539-1545. doi: 10.1002/ptr.5881.\u003c/li\u003e\n\u003cli\u003eKatiyar, D., Sexena, R., Kumar, A., Bansal, P., Prakash, S., Ghosh, D. \u0026amp; Nagarajan, K. (2024). A comprehensive review of the protective effects of herbals against toxicity of Bisphenol A. \u003cem\u003eToxin Reviews\u003c/em\u003e, 43(3), 329-357. doi.org/10.1080/15569543.2024.2329907.\u003c/li\u003e\n\u003cli\u003eKaur, M. \u0026amp; Jindal, R. (2017). Oxidative stress response in liver, kidney and gills of \u003cem\u003eCtenopharyngodon idellus \u003c/em\u003e(cuvier \u0026amp; valenciennes) exposed to chlorpyrifos. \u003cem\u003eMOJ Biology and Medicine,\u003c/em\u003e 1(4), 103-112. doi.10.15406/mojbm.2017.01.00021.\u003c/li\u003e\n\u003cli\u003eKhan, M.H.A. \u0026amp; Parvez, S. (2015). Hesperidin ameliorates heavy metal induced toxicity mediated by oxidative stress in brain of Wistar rats. \u003cem\u003eJournal of Trace Elements in Medicine and Biology,\u003c/em\u003e 31, 53-60. doi: 10.1016/j.jtemb.2015.03.002.\u003c/li\u003e\n\u003cli\u003eKhorasanian, A.S., Fateh, S.T., Gholami, F., Rasaei, N., Gerami, H., Khayyatzadeh, S.S. \u0026amp; Asbaghi, O. (2023). The effects of hesperidin supplementation on cardiovascular risk factors in adults: a systematic review and dose\u0026ndash;response meta-analysis. \u003cem\u003eFrontier in Nutrition,\u003c/em\u003e 10, 1177708. doi: 10.3389/fnut.2023.1177708.\u003c/li\u003e\n\u003cli\u003eKobroob, A., Peerapanyasut, W., Chattipakorn N. \u0026amp; Wongmekiat, O. (2018). Damaging effects of bisphenol A on the kidney and the protection by melatonin: Emerging evidences from \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies. \u003cem\u003eOxidative Medicine and Cellular Longevity,\u003c/em\u003e doi: 10.1155/2018/3082438.\u003c/li\u003e\n\u003cli\u003eKonieczna, A., Rutkowska, A. \u0026amp; Rachoń, D. (2015). Health risk of exposure to bisphenol A (BPA). \u003cem\u003eRocz Panstw Zakl Hig,\u003c/em\u003e 66(1), 5-11.\u003c/li\u003e\n\u003cli\u003eKopjar, N., Žunec, S., Menda\u0026scaron;, G., Micek, V., Ka\u0026scaron;uba, V., Mikolić, A. \u0026amp; Želježić, D. (2018). Evaluation of chlorpyrifos toxicity through a 28-day study: Cholinesterase activity, oxidative stress responses, parent compound/metabolite levels, and primary DNA damage in blood and brain tissue of adult male Wistar rats. \u003cem\u003eChemico- Biological Interaction\u003c/em\u003es, 279, 51-63. doi: 10.1016/j.cbi.2017.10.029. \u003c/li\u003e\n\u003cli\u003eK\u0026uuml;\u0026ccedil;\u0026uuml;kler, S., \u0026Ccedil;omaklı, S., \u0026Ouml;zdemir, S., \u0026Ccedil;ağlayan, C. \u0026amp; Kandemir, F.M. (2021). Hesperidin protects against the chlorpyrifos-induced chronic hepato-renal toxicity in rats associated with oxidative stress, inflammation, apoptosis, autophagy, and up-regulation of PARP-1/VEGF. \u003cem\u003eEnvironmental Toxicology,\u003c/em\u003e 36(8), 1600-1617. https://doi.org/10.1002/tox.23156.\u003c/li\u003e\n\u003cli\u003eLiguori, I., Russo, G., Curcio, F., Bulli, G., Aran, L., Della-Morte, D. \u0026amp; Abete, P. (2018). Oxidative stress, aging, and diseases. \u003cem\u003eClinical Intervention in Aging,\u003c/em\u003e 13, 757-772. doi: 10.2147/CIA.S158513.\u003c/li\u003e\n\u003cli\u003eLiu, T.Y., Wang, C., Han, Y.Z., Bai, C., Ren, H.T., Liu, Y. \u0026amp; Han, X. (2022). Oxidative polymerization of bisphenol A (BPA) via H-abstraction by Bi2.15WO6 and persulfate: Importance of the surface complexes. \u003cem\u003eChemical Engineering Journal,\u003c/em\u003e l 435, 134816. doi: 10.1016/j.cej.2022.134816.\u003c/li\u003e\n\u003cli\u003eLuna LG. (1968). Manual of histologic staining methods of the Armed Forces Institute of Pathology. In Manual of histologic staining methods of the Armed Forces Institute of Pathology (pp. xii-258).\u003c/li\u003e\n\u003cli\u003eManzetti, S., van der Spoel, E.R. \u0026amp; van der Spoel, D. (2014). Chemical properties, environmental fate, and degradation of seven classes of pollutants. \u003cem\u003eChemical Research in Toxicology\u003c/em\u003e 27(5):713-37. doi: 10.1021/tx500014w. \u003c/li\u003e\n\u003cli\u003eMartin Jr J. P., Dailey, M. \u0026amp; Sugarman, E. (1987). Negative and positive assays of superoxide dismutase based on hematoxylin autoxidation. \u003cem\u003eArchives of Biochemistry and Biophysics,\u003c/em\u003e 255(2), 329-36. doi: 10.1016/0003-9861(87)90400-0.\u003c/li\u003e\n\u003cli\u003eMeli, R., Monnolo, A., Annunziata, C., Pirozzi, C. \u0026amp; Ferrante, M.C. (2020). Oxidative stress and BPA toxicity: an antioxidant approach for male and female reproductive dysfunction. \u003cem\u003eAntioxidants,\u003c/em\u003e 9(5), 405. https://doi.org/10.3390/antiox9050405.\u003c/li\u003e\n\u003cli\u003eMohammed, A. (2023). Lycopene attenuates oxidative stress, apoptosis and biochemical fluctuations induced by bisphenol A in the kidneys of rats. \u003cem\u003eEuropean Journal of Anatomy,\u003c/em\u003e 27 (5), 529-540.\u003c/li\u003e\n\u003cli\u003eNasehi, Z., Kheiripour, N., Taheri, M.A., Ardjmand, A., Jozi, F. \u0026amp; Shahaboddin, M.E. (2023). Efficiency of Hesperidin against liver fibrosis Induced by bile duct ligation in rats\u003cem\u003e.\u003c/em\u003e \u003cem\u003eBiomed Research International,\u003c/em\u003e doi\u003cem\u003e: 10.1155/2023/5444301\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eNational Research Council (1996) Guide for the care and use of laboratory animals. Academic Press, Washington.\u003c/li\u003e\n\u003cli\u003eNayak, D., Adiga, D., Khan, N.G., Rai, P.S., Dsouza, H.S., Chakrabarty, S. \u0026amp; Gassman, N.R., Kabekkodu, S.P. (2022). Impact of bisphenol A on structure and function of mitochondria: A Critical Review. \u003cem\u003eReviews Environmental Contamination\u003c/em\u003e (formerly: Residue Reviews), 260, 10. doi.org/10.1007/s44169-022-00011-z.\u003c/li\u003e\n\u003cli\u003eNoshy, P.A., Khalaf, A.A.A., Ibrahim, M.A., Mekkawy, A.M., Abdelrahman,, R.E., Farghali, A., Tammam, A.A. \u0026amp; Zaki, A.R. (2022). Alterations in reproductive parameters and steroid biosynthesis induced by nickel oxide nanoparticles in male rats. The ameliorative effect of hesperidin. \u003cem\u003eToxicology,\u003c/em\u003e 473, 153208.doi.10.1016/j.tox.2022.153208. \u003c/li\u003e\n\u003cli\u003eOlujimi, O., Ayoola, R., Olayinka, O., Dosumu, O., Rotimi, S. \u0026amp; Aladesida, A. (2020). Evaluation of antioxidant enzymes performances and DNA damage induced by bisphenol A and diisobutylphthalate in \u003cem\u003eHyperiodrilus africanus\u003c/em\u003e-earthworms. \u003cem\u003eEmerging Contaminants,\u003c/em\u003e 6, 1-9. doi.10.1016/j.emcon.2019.10.001.\u003c/li\u003e\n\u003cli\u003eOmodon, A.C., Onwuka, O.M., Adele, B.O. \u0026amp; Ige, A.O. (2024). cardiotoxic effects of Bisphenol A in male wistar rats are attenuated by Garcinia kola and its biflavonoid, kolaviron, via antioxidant and antiinflammation-based mechanisms. \u003cem\u003eJournal of Traditional and Complementary Medicine\u003c/em\u003e, doi.org/10.1016/j.jtcme.2024.08.004.\u003c/li\u003e\n\u003cli\u003eOsama, H.M., Khadrawy, S.M., El-Nahass, E., Othman, S.I. \u0026amp; Mohamed, H.M. (2024). Eltroxin and Hesperidin mitigate testicular and renal damage in hypothyroid rats: amelioration of oxidative stress through PPAR\u0026gamma; and Nrf2/HO-1 signaling pathway. \u003cem\u003eLaboratory Animal Research\u003c/em\u003e, 40, 19. doi.org/10.1186/s42826-024-00204-8.\u003c/li\u003e\n\u003cli\u003eOwumi, S.E. \u0026amp; Dim, U.J. (2019). Manganese suppresses oxidative stress, inflammation and caspase-3 activation in rats exposed to chlorpyrifos. \u003cem\u003eToxicology Reports,\u003c/em\u003e 6, 202-209. doi: 10.1016/j.toxrep.2019.02.007.\u003c/li\u003e\n\u003cli\u003ePaglia, D.E. \u0026amp; Valentine, W.N. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. \u003cem\u003eJournal of Laboratory and Clinical Medicine,\u003c/em\u003e 70(1), 158-169.\u003c/li\u003e\n\u003cli\u003ePari, L., Karthikeyan, A., Karthika, P. \u0026amp; Rathinam, A. (2015). Protective effects of hesperidin on oxidative stress, dyslipidaemia and histological changes in iron-induced hepatic and renal toxicity in rats. \u003cem\u003eToxicology Reports,\u003c/em\u003e 2, 46-55. doi: 10.1016/j.toxrep.2014.11.003.\u003c/li\u003e\n\u003cli\u003ePelletier, G. Wang, G.S., Wawrzynczak, A., Rigden, M., Aranda-Rodriguez, R. \u0026amp; Caldwell, D. (2026). Direct Comparison of the Impacts of Bisphenol A, Bisphenol F, and Bisphenol S in a Male Rat 28-Day Oral Exposure Study. \u003cem\u003eInternational Journal of Toxicology,\u003c/em\u003e 45(1), 4\u0026ndash;21. doi: 10.1177/10915818251378990. \u003c/li\u003e\n\u003cli\u003ePyrzynska, K. (2022). Hesperidin: A review on extraction methods, stability and biological activities. \u003cem\u003eNutrients,\u003c/em\u003e 14(12), 2387. https://doi.org/10.3390/nu14122387.\u003c/li\u003e\n\u003cli\u003eRather, I.A., Koh, W.Y., Paek, W.K. \u0026amp; Lim, J. (2017). The sources of chemical contaminants in food and their health implications. \u003cem\u003eFrontier in Pharmacology,\u003c/em\u003e 17, 8:830. doi: 10.3389/fphar.2017.00830.\u003c/li\u003e\n\u003cli\u003eSakinah, E.N., Wisudanti, D.D., Abrori, C., Supangat, S., Ramadhani, L.R., Putri, I.S., Pamungkas, G.C., Arrobani, M.H., Rahmadina, R. \u0026amp; Dirgantara, W. (2024). The effect of chlorpyrifos oral exposure on the histomorphometric and kidney function in Wistar rat. \u003cem\u003eIndian Journal Pharmacology\u003c/em\u003e, 56, 186-90.\u003c/li\u003e\n\u003cli\u003eSaoudi, M., Badraoui, R., Rahmouni, F., Jamoussi, K. \u0026amp; El Feki, A. (2021). Antioxidant and protective effects of artemisia campestris essential oil against chlorpyrifos-induced kidney and liver injuries in rats. \u003cem\u003eFrontier in Physiology, \u003c/em\u003e12 \u0026ndash; 2021. https://doi.org/10.3389/fphys.2021.618582.\u003c/li\u003e\n\u003cli\u003eSteffensen, I.L., Dirven, H., Couderq, S., David, A., D\u0026apos;Cruz, S.C., Fern\u0026aacute;ndez, M.F., Mustieles, V., Rodr\u0026iacute;guez-Carrillo, A. \u0026amp; Hofer, T. (2020). Bisphenols and oxidative stress biomarkers-associations found in human studies, evaluation of methods used, and strengths and weaknesses of the biomarkers. \u003cem\u003eInternational Journal of Environmental Research and Public Health,\u003c/em\u003e 17(10), 3609. doi: 10.3390/ijerph17103609.\u003c/li\u003e\n\u003cli\u003eSule, R.O., Condon, L. \u0026amp; Gomes, A.V. (2022). A common feature of pesticides: oxidative stress\u0026mdash;the role of oxidative stress in pesticide-induced toxicity. \u003cem\u003eOxidative Medicine and Cellular Longevity,\u003c/em\u003e doi: 10.1155/2022/5563759.\u003c/li\u003e\n\u003cli\u003eTabeshpour, J., Hosseinzadeh, H., Hashemzaei, M. \u0026amp; Karimi, G. (2020). A review of the hepatoprotective effects of hesperidin, a flavanon glycoside in citrus fruits, against natural and chemical toxicities. \u003cem\u003eDaru\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003eJournal of Pharmaceutical Sciences,\u003c/em\u003e 28(1), 305-317. doi: 10.1007/s40199-020-00344-x.\u003c/li\u003e\n\u003cli\u003eTsen, C.M., Liu, J.H., Yang, D.P., Chao, H.R., Chen, J.L., Chou, W.C. \u0026amp; Chuang, C.Y. (2021). Study on the correlation of bisphenol A exposure, pro-inflammatory gene expression, and C-reactive protein with potential cardiovascular disease symptoms in young adults. \u003cem\u003eEnvironmental Science and Pollution Research International,\u003c/em\u003e doi: 10.1007/s11356-021-12805-0. \u003c/li\u003e\n\u003cli\u003eUchendu, C., Ambali, S.F., Ayo, J.O. \u0026amp; Esievo, K.A.N. (2015). The protective role of alpha-lipoic acid on long-term exposure of rats to the combination of chlorpyrifos and deltamethrin pesticides. \u003cem\u003eToxicology and Industrial Health,\u003c/em\u003e 31(12), 1061-1347.doi.org/10.1177/0748233715616553.\u003c/li\u003e\n\u003cli\u003eUchendu, C., Ambali, S.F., Ayo, J.O., Esievo, K.A.N. \u0026amp; Umosen, A.J. (2014). Erythrocyte osmotic fragility and lipid peroxidation following chronic co-exposure of rats to chlorpyrifos and deltamethrin, and the beneficial effect of alpha-lipoic acid. \u003cem\u003eToxicology Reports,\u003c/em\u003e 1, 373-378. doi.org/10.1016/j.toxrep.2014.07.002.\u003c/li\u003e\n\u003cli\u003eVandenberg, L.N., Ehrlich, S., Belcher, S.M., Ben-Jonathan, N., Dolinoy, D.C., Hugo, E.R., Hunt, P.A., Newbold, R.R., Rubin, B.S., Sail, K.S., Soto, A.M., Wang, H. \u0026amp; vom Saal, F.S. (2013). Low dose effects of bisphenol A an integrated review of in vitro, laboratory animal, and epidemiology studies. \u003cem\u003eEndocrine Disruptors,\u003c/em\u003e 1, 1, e25078. doi: 10.4161/endo.26490.\u003c/li\u003e\n\u003cli\u003eWołejko, E., Łozowicka, B., Jabłońska-Trypuć, A., Pietruszyńska, M. \u0026amp; Wydro, U. (2022). Chlorpyrifos occurrence and toxicological risk assessment: a review. \u003cem\u003eInternational Journal of Environmental Research and Public Health, \u003c/em\u003e19 (19), 12209. doi: 10.3390/ijerph191912209.\u003c/li\u003e\n\u003cli\u003eWu, X., Xie, W., Cheng, Y. \u0026amp; Guan, Q. (2016). Severity and prognosis of acute organophosphorus pesticide poisoning are indicated by C-reactive protein and copeptin levels and APACHE II score. \u003cem\u003eExperimental and Therapeutic Medicine,\u003c/em\u003e 11(3), 806-810. doi: 10.3892/etm.2016.2982. \u003c/li\u003e\n\u003cli\u003eWu, Y., Chang, S., Chen, H., Tsai, K., Lee, W., Wang, I., Lee, C., Chen, C., Liu, S., Weng, C., Huang, W., Hsu, C. \u0026amp; Yen, T. (2023). Human poisoning with chlorpyrifos and cypermethrin pesticide mixture: Assessment of clinical outcome of cases admitted in a tertiary care hospital in Taiwan. \u003cem\u003eInternational Journal of General Medicine,\u003c/em\u003e 16, 4795-4804. doi: 10.2147/IJGM.S432861. \u003c/li\u003e\n\u003cli\u003eYaqub, S.A., Rahamon, S.K. \u0026amp; Arinola, O.G. (2023). Serum levels of selected inflammatory markers in farm workers exposed to organophosphate pesticides. \u003cem\u003eAfrican Journal of Biomedical Research\u003c/em\u003e 26(1): 89-93.\u003c/li\u003e\n\u003cli\u003eYavuz, T., Delibas, N., Yildirim, B., Altuntas, I., Candır, O., Cora, A. \u0026amp; Kutsal, A. (2004). Vascular wall damage in rats induced by methidathion and ameliorating effect of vitamins E and C. \u003cem\u003eArchives of Toxicology,\u003c/em\u003e 78(11), 655-9. doi: 10.1007/s00204-004-0593-9.\u003c/li\u003e\n\u003cli\u003eZhang, Y., Jia, Q., Hu, C., Han, M., Guo, Q., Li, S. \u0026amp; Peng, C. (2021). Effects of chlorpyrifos exposure on liver inflammation and intestinal flora structure in mice. \u003cem\u003eToxicology Research\u003c/em\u003e (Camb), 10(1), 141-149. doi: 10.1093/toxres/tfaa108.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Effect of chronic exposure to corn oil (C/oil), chlorpyrifos (CPF), bisphenol A (BPA), chlorpyrifos and bisphenol A (CPF+BPA), hesperidin (HES) and chlorpyrifos, bisphenol A and hesperidin (CPF+BPA+HES) on serum oxidative stress biomarkers in Wistar rats.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"670\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003eC/oil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eCPF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eBPA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eCPF+BPA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003eHES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003eCPF+BPA+HES\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eSOD (IUL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e3.55\u0026plusmn;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e2.05\u0026plusmn;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2.55\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e2.18\u0026plusmn;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e4.37\u0026plusmn;0.13\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e3.2\u0026plusmn;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eCAT\u003c/p\u003e\n \u003cp\u003e(IUL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e9.83\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e2.68\u0026plusmn;0.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e5.8\u0026plusmn;0.40\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e2.75\u0026plusmn;0.61\u003csup\u003eac\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e8.8\u0026plusmn;0.62\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e6.03\u0026plusmn;0.56\u003csup\u003eabde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eGPx\u003c/p\u003e\n \u003cp\u003e(IUL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.17\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e0.36\u0026plusmn;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.9\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.68\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e1.53\u0026plusmn;0.32\u003csup\u003ebd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e1.1\u0026plusmn;0.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003cp\u003e(nmolml\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e3.9\u0026plusmn;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e10.02\u0026plusmn;1.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e5.1\u0026plusmn;0.23\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e6.17\u0026plusmn;0.87\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e2.6\u0026plusmn;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e4.16\u0026plusmn;0.68\u003csup\u003ebd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eP \u0026lt; 0.05 compared to C/oil group\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eP \u0026lt; 0.05 compared to CPF group\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003eP\u0026lt; 0.05 compared to BPA group\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ed\u003c/sup\u003eP \u0026lt; 0.05 compared to CPF+BPA group\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ee\u003c/sup\u003eP \u0026lt; 0.05 compared to HES group\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bisphenol A, Chlorpyrifos, Oxidative stress, Hesperidin, Antioxidant","lastPublishedDoi":"10.21203/rs.3.rs-9163020/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9163020/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: The extensive use of pesticides and the increasing production and consumption of bisphenol A found in plastics pose new challenges regarding toxicity and environmental pollution. The study aimed to evaluate the protective effects of hesperidin on hepato-renal and oxidative stress changes provoked by chronic co-exposure of rats to bisphenol A (BPA) and chlorpyrifos (CPF). Thirty male Wistar rats were assigned into six groups of five rats each: Group I (C/oil) were administered Corn oil (2 mlkg\u003csup\u003e-1\u003c/sup\u003e), II (CPF) and III (BPA) were administered chlorpyrifos (4.75mgkg\u003csup\u003e-1\u003c/sup\u003e; 1/20\u003csup\u003eth\u003c/sup\u003e LD\u003csub\u003e50\u003c/sub\u003e) and bisphenol A (LOAEL; 50 mgkg\u003csup\u003e-1\u003c/sup\u003eday\u003csup\u003e-1\u003c/sup\u003e), respectively, while group IV (CPF+BPA) was co-exposed to CPF (4.75 mgkg\u003csup\u003e-1\u003c/sup\u003e) and BPA (50mgkg\u003csup\u003e-1\u003c/sup\u003eday\u003csup\u003e-1\u003c/sup\u003e). Group V (HES) was administered hesperidin (100 mgkg\u003csup\u003e-1\u003c/sup\u003e), while group VI (CPF+BPA+HES) rats were pretreated with HES (100mgkg\u003csup\u003e-1\u003c/sup\u003e) and then co-exposed to CPF (4.75mgkg\u003csup\u003e-1\u003c/sup\u003e) and BPA (50mgkg\u003csup\u003e-1\u003c/sup\u003eday\u003csup\u003e-1\u003c/sup\u003e) 30 minutes later. The different treatments were administered orally once daily for 16 weeks. At the end of the experiment, blood samples were collected and the serum was used to assess various health markers. These included total protein, albumin, urea, creatinine, and C-reactive protein levels, as well as the activity of certain enzymes like aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). Additionally, the levels of antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured, along with the levels of malondialdehyde (MDA). The liver and kidney tissues were also examined for any signs of damage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The results showed that exposure to both BPA and CPF caused significant changes in these biochemical parameters and damaged the liver and kidneys. However, supplementation with HES helped to reduce the harmful effects of co-exposure to BPA and CPF, suggesting a potential protective role.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Low dose of the combination of the CPF and BPA resulted in marked alterations in the parameters evaluated, and hesperidin offered some level of protections to the liver and kidneys of exposed rats. This protective effects may be due to its anti-inflammatory and the antioxidant ability to scavenge reactive oxygen species generated by the contaminants.\u003c/p\u003e","manuscriptTitle":"Chronic Hepatorenal Toxicity Induced by Low-Dose Co-Exposure to Chlorpyrifos and Bisphenol A in Wistar Rats: Protective Effects of Hesperidin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 13:55:54","doi":"10.21203/rs.3.rs-9163020/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"67debd12-2934-4f3f-9ce0-aabbebe0a530","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-18T09:09:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T15:49:05+00:00","index":21,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T11:08:26+00:00","index":20,"fulltext":""},{"type":"reviewerAgreed","content":"260575233978175026973242687915700960322","date":"2026-05-07T10:28:47+00:00","index":19,"fulltext":""},{"type":"reviewerAgreed","content":"225840002971097347029205563120071971279","date":"2026-05-06T10:07:17+00:00","index":18,"fulltext":""},{"type":"reviewerAgreed","content":"241231268444476347790154022958891084552","date":"2026-04-29T14:00:04+00:00","index":17,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T09:25:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 13:55:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9163020","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9163020","identity":"rs-9163020","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}