Hesperetin and naringenin relieve neurotoxicity and histological distortions in benzo[a]pyrene-challenged rats

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Maduako, Chidinma N. Igwe, Chinweokwu V. Ego, Osarhieme T. Okugbo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6706130/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 Introduction: Environmental pollutants like benzo[a]pyrene (BaP), which belongs to the PAH family, generated from fossil fuel combustion, pose a neurotoxic risk. Their lipophilic nature allows them to breach the blood-brain barrier and trigger oxidative stress. This research explored the neuroprotective effectiveness of the flavonoids hesperetin together with naringenin against BaP-induced neurotoxicity in a rodent model. Methods A total of 28 male Wistar rats were randomly distributed into four experimental groups and treated with BaP alone or in combination with hesperetin or naringenin for 14 days. Results The results demonstrated that BaP exposure led to significant depletion of glutathione, inhibition of glutathione peroxidase, and elevation of lipid peroxidation, hydrogen peroxide, and xanthine oxidase activity in rat brain tissue. Histopathological analysis revealed neuronal damage characterized by pyknotic cells in the cerebrum and shrunken Purkinje cells in the cerebellum. Co-administration of hesperetin markedly attenuated these oxidative stress markers and prevented histopathological alterations. Naringenin also exhibited some protective effects against structural damage. Discussion Our findings suggest that hesperetin and naringenin can mitigate BaP-induced neurotoxicity by bolstering antioxidant defenses and preserving brain tissue integrity. These results highlight the potential neuroprotective benefits of flavonoid-rich citrus fruits in combating the adverse effects of environmental pollutants like BaP. Applied Biochemistry Benzo[a]pyrene Hesperetin Naringenin Neurotoxicity chemoprevention Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Civilization's progress is intertwined with industrialization and urbanization, leading to increased reliance on fossil fuels for energy production. The combustion of these fuels, such as coal and oil, generates polycyclic aromatic hydrocarbons (PAHs) as byproducts. These environmental contaminants, including benzo[a]pyrene (BaP), are widespread due to extensive industrial activities [ 1 ]. PAHs can persist in the environment, adhering to particles of various sizes and accumulating in organisms. While individual PAHs may not be definitively classified as human carcinogens, PAH mixtures are recognized as cancer-causing agents, particularly in occupations with high exposure, such as coal mining and soot-related industries [ 2 – 4 ]. BaP, a classical compound for investigating the impact of this type of PAH. Currently, neurological disorders are on the rise. In the US for example, an estimated 1.1 million adults develop brain disorders annually, with over 13 million individuals living with these conditions [ 5 ]. Also, neurological disorders are becoming more common in Sub-Saharan Africa and this increase is linked to factors such as rising vehicular traffic, alcohol and substance abuse [ 6 ]. The economic burden of mental illness is substantial, with an estimated €810 billion spent in Europe in 2010 and a global cost of $ 604 billion for dementia in the same year [ 7 ]. These figures underscore the critical need for research and management strategies to address this growing health concern. The brain, with its high metabolic activity, demands a constant supply of oxygen and glucose. This metabolic intensity makes it vulnerable to oxidative stress, leading to lipid peroxidation (due to its rich lipid content), disruption of signaling pathways, and dysregulation of gene expression [ 8 ]. The brain's limited regenerative capacity and moderate antioxidant defenses further exacerbate its susceptibility to reactive oxygen species (ROS)-mediated damage [ 9 ]. Oxidative stress has been implicated in the pathogenesis of various neurological disorders, including Parkinson's disease, Alzheimer's disease, and schizophrenia, through mechanisms involving excitotoxicity, apoptosis, and cellular signaling alterations [ 10 ]. While DNA adduct formation is a well-established mechanism of PAH carcinogenicity, oxidative stress playing a crucial role. PAHs can induce redox cycling, leading to ROS generation and overwhelming endogenous antioxidant systems [ 1 ]. The lipophilic nature of PAHs enables their penetration of the blood-brain barrier, facilitating their entry into brain tissue and exerting their deleterious effects [ 2 ]. Antioxidant compounds offer a promising avenue for mitigating oxidative stress. These compounds can scavenge free radicals, enhance antioxidant enzyme activity, chelate metal ions, and boost endogenous antioxidant levels [ 11 , 12 ]. Flavonoids, a class of polyphenolic phytochemicals, exhibit potent antioxidant properties due to their numerous hydroxyl and methoxy groups [ 11 ]. Furthermore, flavonoids demonstrate regulatory effects on various cellular processes, including inflammation and carcinogenesis, by modulating gene expression [ 13 ]. This evidence has spurred the investigation of compounds like curcumin, resveratrol, and lycopene in clinical trials for various cancers [ 13 ]. Hesperetin (5,7,3′-trihydroxy-4′-methoxyflavanone), a flavanone abundant in citrus fruits, particularly oranges, is obtained from hesperidin through deglycosylation [ 14 ]. Studies have demonstrated hesperetin's potential in inhibiting cell proliferation, promoting apoptosis, and suppressing angiogenesis in rat colon carcinogenesis models [ 15 , 16 ]. In human colon adenocarcinoma cells (HT-29), hesperetin has been shown to inhibit cell growth, downregulate tumor markers, and upregulate Notch1 mRNA and protein levels [ 17 ]. While Naringenin, chemically known as 5,7-dihydroxy-2-(4-hydroxyphenyl) chroman-4-one, an flavonone primarily found in grapefruit [ 18 ], exhibits various biological activities. These include adipogenesis inhibition, reduced insulin sensitivity [ 19 ], suppression of atopic dermatitis in mice [ 19 ], protection against carbon tetrachloride-induced nephrotoxicity in mice [ 20 ], and amelioration of hyperglycemia-mediated inflammation in experimental diabetic rats [ 21 ]. This research examined how oxidative stress contributes to the neurotoxic impact of BaP on rat brain tissue and evaluated the neuroprotective effects of Hesperetin and Naringenin against oxidative injury caused by BaP. 2. Materials and methods 2.1. Chemicals and reagents Benzo[a]pyrene (> 95%) was acquired from Sigma-Aldrich, Missouri, USA. Hesperetin (98%) and Naringenin (98%) were procured from AK Scientific, Union City, California, USA. All other chemicals utilized in this research were of analytical grade, obtained from Sigma-Aldrich, located in St. Louis, Missouri, USA, and British Drug Houses in Poole, Dorset, UK. 2.2. Animals Thirty-two adult Wistar strain male albino rodents (180–200g) were procured from the Animal House, Department of Medical Biochemistry. The animals were housed in ventilated cages under controlled temperature conditions (22 ± 2°C) and a standardized 12-hour light/dark cycle. The animals were provided with unrestricted availability of feed and drinking water. After adapting to their environment for 7 days, the animals were arbitrarily assigned to one of four separate cohorts, each comprising seven animals. All experimental protocols were carried out following the guidelines of the Benson Idahosa University Ethics Committee Animal Handling Protocol (ECBIUAHP-0012/med/089). 2.3. Study design The animals were grouped and treated as below: CTRL CORN OIL (2.5 ml/kg) BaP BENZO[A]PYRENE (4 mg/kg) BaP + NAR BENZO[A]PYRENE (4 mg/kg) + NARINGENIN (50 mg/kg) BaP + HPN BENZO[A]PYRENE (4 mg/kg) + HESPERETIN (50 mg/kg) Treatment was performed via oral gavage daily for 28 consecutive days. The dose of BaP, Hesperetin and Naringenin were selected based on previous studies [ 22 – 24 ]. Benzo[a]pyrene was slightly solubilized in dimethylsulfoxide and mixed in corn oil, while Naringenin and Hesperetin were dissolved in corn oil. Twenty-four hours following the final treatment, the subjects were euthanized using the cervical dislocation method. Brains were promptly removed and rinsed in chilled potassium chloride buffer. For biochemical analyses, brain tissues were weighed, blended in chilled phosphate-buffered solution (pH 7.4) at a 1:4 weight-to-volume ratio using a glass homogenizer. Centrifugation of the homogenates was performed at 10,000 g for 15 minutes at 4°C. The resulting supernatants, following the mitochondrial phase, were kept at -80°C until further investigation. For microscopic tissue assessment, neural samples were preserved in 10% formaldehyde for subsequent processing and embedding. 2.4. Biochemical analyses 2.4.1. Assessment of neuro-anti-oxidative biomarkers Catalase activity in the brain was measured by monitoring the decrease in absorbance at 240 nm as hydrogen peroxide is consumed, according to the method described by Claiborne [ 25 , 26 ]. Hydrogen peroxide concentration, which correlates with absorbance at this wavelength, was quantified by applying an extinction coefficient value of 0.0436 mM⁻¹cm⁻¹. SOD activity was evaluated using the procedure described by Misra and Fridovich [ 27 ], which measures the inhibition involving adrenaline autoxidation to its oxidation product at pH 10.2. Lipid peroxidation was quantified using the Varshney and Kale method, modified by Farombi [ 28 , 29 ]. TBARS (thiobarbituric acid reactive compounds) and MDA (malondialdehyde) were determined, with MDA forming a pink complex with 2-thiobarbituric acid under acidic conditions, detectable at 532 nm. GST (Glutathione S-transferase) activity was assessed following the procedure outlined by Habig [ 30 ]. The enzyme function was evaluated through the coupling of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione. This shift in the absorbance spectrum to 340 nm allows for direct measurement of enzyme activity [ 26 , 29 ], while the concentration of reduced glutathione (GSH) was measured using the procedure described by Beutler [ 31 ]. Glutathione levels were measured using Ellman's reagent 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB). DTNB reacts with sulfhydryl groups, producing a yellow product with maximum absorbance at 412 nm [ 32 ]. Xanthine oxidase (XO) activity was assessed using the method of Bergmeyer [ 33 ]. The total sulfhydryl group (-TSH) in biological sample was estimated according to Ellmans [ 34 ]. It relies Ellmans reagent reaction with sulfhydryl groups to produce a yellow coloured complex. Neuro assessment of pro-inflammatory markers Myeloperoxidase (MPO) activity was determined using a previously described method [ 35 ] Levels of nitric oxide (NO) were determined by assessing nitrite concentrations using a Griess reagent according to a standard protocol [ 36 ]. Concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) were quantified in the brain using commercial ELISA kits from Elabscience (China) and analyzed on a SpectraMax microplate reader from Molecular Devices (USA) following their guidelines. Monitoring of reactive oxygen and nitrogen species (RONS) production in brain tissue Reactive oxygen and nitrogen intermediates (RONS) production was assessed within brain tissues using a previously described method [ 37 ]. This relies on oxidising 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) into 2',7'-dichlorofluorescein (DCF) by tissue-derived RONS. Succinctly, 10 µL of the tissue homogenate was mixed with 5 µL of DCFH-DA (200 µM; achieving a final concentration of 5 µM) in 150 µL of 0.1M potassium phosphate buffer (pH 7.4) along with 35 µL of distilled water. After DCFH-DA oxidation, the fluorescence signal of DCF was detected at excitation and emission wavelengths of 488 nm and 525 nm, respectively, using a SpectraMax microplate reader. The DCF generation rate was a percentage relative to the control group. Examination of Tissue Morphology Brain samples were preserved in 10% formalin for 7 days. Following fixation, tissues were processed for paraffin embedding. This involved serial dehydration in graded ethanol solutions, followed by clearing in xylene. Tissues embedded in paraffin were sliced into 5 µm thick sections with a microtome. The tissue sections were placed on glass slides, cleared of paraffin using xylene, and gradually rehydrated through a series of graded ethanol solutions. Afterward, the sections were stained with hematoxylin and eosin (H&E) and observed under a light microscope. Microscopic tissue evaluation was carried out following the procedures described by Hotchkis and McManus [ 38 ]. Statistical analysis The data were subjected to one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test for multiple comparisons, utilizing GraphPad PRISM 8 software. A p-value of less than 0.05 was considered statistically significant. Results are expressed as mean ± standard error of the mean (SEM). 3. Results NAR + HPN improve neurotoxicity in BaP-challenged rats ( Fig. 1 A & B) , Antioxidant enzyme and glutathione levels: BaP exposure caused a marked (p < 0.05) decline in Glutathione S-transferase (GST) activities, as well as in the concentration of reduced Glutathione (GSH) compared to the control group. Combined treatment with NAR + HPN dosed at 50 mg/kg each reversed the BaP-induced decreases, restoring GST, and GSH levels. (Fig. 1 C, D & E) : antioxidant enzyme activities and total sulfhydryl group (TSH) levels: Similarly, BaP exposure led to a marked (p < 0.05) reduction in CAT and SOD activities, as well as a reduction in TSH levels, compared to the control group. NAR + HPN co-administration significantly attenuated these BaP-induced reductions in CAT, SOD, and TSH. Figure 1 F, G & H) : oxidative stress markers and xanthine oxidase (XO) activity: BaP administration markedly (p < 0.05) elevated the concentrations of lipid peroxidation (LPO), reactive oxygen and nitrogen intermediates, as well as XO activity in rat brain, relative to the control group. Co-administration of NAR + HPN significantly reduced (p < 0.05) these BaP-induced elevations in LPO, RONS, and XO activity. NAR + HPN diminishes BaP-mediated increase in inflammatory indices in the brain of rats The NAR + HPN effect against BaP-instigated increase in the indices of inflammation is presented in Fig. 2 . BaP treatment alone increased (p < 0.05) NO level and MPO activity in rats brain relative to control. These mediated increases were reduced (p < 0.05) when the rats were administered with NAR + HPN. Figure 3 : Exposure to BaP led to a marked increase (p < 0.05) in levels of the proinflammatory mediators TNF-α and IL-1β within the brains of rats relative to the control group. Such increases were notably reduced following NAR + HPN administration. NAR + HPN abrogated histological alterations in rats brain following BaP challenge The photomicrographs of brain from rats exposed to BaP and NAR + HPN were examined microscopically in (Fig. 4 A, B & C). The images observed from the control and NAR + HPN-treated rats were consistent with intact brain morphological and histological architecture, while the group challenged with BaP presented altered histological architecture. 4. Discussions This investigation aimed to evaluate the impact of Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), on brain antioxidant capacity and structural integrity. A 28-day BaP administration protocol at 2 mg/kg, established by Maciel et al. [ 22 ], was employed to mimic exposure levels observed in individuals with high consumption of grilled foods. The dosing regimen complied with the standards set by the Organization for Economic Co-operation and Development (OECD) for repeated-dose toxicity assessments. Other studies have utilized higher BaP doses (25–200 mg/kg) in single-dose exposure models to mimic specific scenarios like occupational exposures [ 39 ]. Approximately 40% of orally administered BaP is bioavailable, resulting in a relatively low effective concentration at target tissues [ 39 ]. Furthermore, we hypothesized that an intervention strategy involving natural products such as Naringenin (NAR) and Hesperetin (HPN) could mitigate BaP-induced neurotoxicity. The toxic effect of BaP is linked to its highly reactive intermediate and the production of reactive oxygen species (ROS) during its bioactivation and can induce oxidative stress, initiate DNA damage and disrupt cellular activities [ 40 , 41 ]. We investigate strategies to mitigate BaP neurotoxicity, which is attributed to compromised antioxidant defenses, oxido-inflammatory responses and the formation of reactive oxygen species [ 42 ]. Our approach involves the development of preventive strategies, including the use of plant-derived natural products to counteract BaP-induced neurotoxicity and promote neuronal health [ 42 ]. SOD and CAT represent key antioxidant enzymes that act cooperatively to neutralize RONs. SOD facilitates the conversion of superoxide ions (O 2 - ) to hydrogen peroxide, thereby reducing oxidative stress. Subsequently, CAT decomposes H 2 O 2 into water and oxygen, completing the detoxification process and maintaining cellular redox balance. The administration of BaP to rats in the study markedly diminished SOD and CAT activities, indicating enzyme inhibition, thus accumulation of toxic superoxide radicals and hydrogen peroxide molecules in the brain, causing oxidative stress. However, co-administration of NAR and HPN to rats challenged with BaP, significantly enhanced SOD and CAT activities, suggesting NAR and HPN antioxidant capacity, which may mitigate BaP induced neurotoxicity. Oxidative stress occurs when the delicate balance between antioxidants and pro-oxidants is disrupted, favouring the dominance of pro-oxidants. This imbalance allows ROS to accumulate, causing cellular damage [ 43 ]. Free radicals generated from oxidative stress can undermine both enzymatic and non-enzymatic cellular antioxidant defenses, leading to macromolecular damage, enhanced lipid peroxidation, and increased reactive oxygen and nitrogen species levels [ 44 ]. Elevated RONS production plays a significant role in triggering and advancing injuries as well as many systemic disorders [ 45 ]. Elevated levels of RONS, LPO, and XO activity mark the characteristic profile of oxidative stress. Conversely, this imbalance is often accompanied by decreased activity of the enzymatic antioxidant GST and diminished concentration of GSH and total thiol TSH, indicating compromised cellular antioxidant defenses. Our study reveals that exposure to BaP alone led to significant decline in brain GSH and TSH levels, accompanied by increases in LPO, RONS levels, and xanthine oxidase activity. These findings suggest that BaP-induced oxidative stress can cause neuronal toxicity, which is consistent with [ 46 ]. However, co-administration of NAR and HPN significantly enhanced GST activity and restored GSH and TSH levels, indicating their potential neuroprotective impact in response to BaP-stimulated oxidative stress. Similarly, the combined administration of NAR and HPN notably lowered LPO, RONS, and XO activity in the treated rats, demonstrating antioxidant characteristics. The observed outcomes associated with NAR and HPN are likely due to their inherent antioxidant properties [ 21 – 24 ]. Further, nitric oxide is capable of interacting with superoxide anion (O 2 -), leading to the generation of peroxynitrite (ONOO-), a deleterious RNS, while MPO, a heme-containing enzyme primarily released by neutrophils is closely linked to the development of oxidative stress and inflammation. However, unabated degranulation exacerbates inflammation, and tissue damage ensues [ 47 , 48 ]. The proinflammatory cytokines, IL-1β and TNF-α, are star players during inflammatory processes, facilitating cell recruitment to injury sites [ 49 ]. Elevated concentrations of IL-1β and TNF-α in BaP-exposed rats brains indicate a pronounced inflammatory response. Increased IL-1β and TNF-α can, in turn, activate iNOS leading to overproduction of NO perpetuate oxidative stress and tissue anomaly [ 50 ]. Our findings are consistent with previously reported data on BaP-induced changes in inflammation bioamarkers [ 51 , 52 ]. The elevated levels of neuronal NO, IL-1β and TNF-α, as well as increased MPO activity, observed in BaP-teated rats, suggest that BaP exposure may lead to neuronal dysfunction via inflammatory and nitrosative stress. In contrast, treatment with NAR and HPN mediated significant reductions in NO, IL-1β, TNF-α levels, and MPO activity in BaP-challenged rats, demonstrating the anti-inflammatory effects of NAR and HPN against BaP-induced neuroinflammatory responses. The histopathological assessment provides a comprehensive evaluation of tissue architecture and disease progression, visually confirming the biochemical alterations observed in the study. Our findings revealed that administration of BaP alone caused pronounced degeneration of cortical neurons and Purkinje neurons (PNCA3) in treated rats. Furthermore, histomorphometric analysis demonstrated significant reductions in the densities of cortical neurons, PNCA3, granule cell layer of the dentate gyrus (GCDG), and white matter of the posterior cerebral cortex (WDPC) [ 53 ]. These histopathological and histomorphometric alterations observed in the brains of rats treated with BaP are likely associated with oxidative damage and neuronal inflammation. Our observations align with previous study of Farombi et al [ 54 ]. Cotreatments with NAR and HPN as we observed, reversed these histological and morphometrical changes, preserving brain structures and neurons in a manner comparable to the control. This finding aligns with biochemical effects of NAR and HPN against BaP-induced neurotoxicity in rats. 5. Conclusion The present data suggest that NAR and HPN effectively mitigated neuronal toxicity in BaP-treated mice via multiple biochemical mechanisms. They also protected against BaP-induced inflammation and supported antioxidant defenses. The study's observed health benefits of NAR and HPN highlight their potential as therapeutic phytochemicals for addressing BaP-induced brain toxicity. Declarations Funding This study received no funding from either public, private or commercial bodies. CRediT authorship contribution Ikenna C. Maduako : Conceptualisation, Methodology, Data curation, writing- original draft. Chidinma N. Igwe : writing original draft. Chinweokwu V. Ego: Project administration and proofreading the original draft. Uzoamaka N. Ngwoke: Proofreading the original draft. Abraham O. Ekhegbesela : data curation. Esther A. Omoyajowo : data curation, writing original draft Conflicting interest The authors have no conflicts to declare Acknowledgements The authors thank Dr. Iramofu Dominic of Georgia Institute of Technology, USA, for the gift of Benzo[a]pyrene used in this study and the University of Buea, Cameroon. 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Afr J Med Med Sci 36:103–108 Farombi EO, Awogbindin IO, Owoeye O, Maduako IC, Ajeleti AO, Owumi SE (2020) Kolaviron via anti-inflammatory and redox regulatory mechanisms abates multi-walled carbon nanotubes-induced neurobehavioral deficits in rats. Psychopharmacology. 10.1007/s00213-019-05432-8 Additional Declarations The authors declare no competing interests. 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6706130","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":459222114,"identity":"df3fa471-2dab-466b-9a90-a2fe3985ec05","order_by":0,"name":"Ikenna C. Maduako","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBACNiCWYGBIYOCXYGwAsplJ0CI5g1gtDDAtBjfAbCK08IkdfnibpyZNzvh2c5sEQ4V1YoN0+wX8DpNOM7bmOZZjbHbnIFDLmfTEBpkzBQS0JJhJ87BVJG67kdgmwdh2OLFBIieBgJb0b9I8/yoSN88AaflHlJYcM2netpzEDRIgLQ0gLekHCGkptpzbl2Yscedgs0XCsXTjNokcvDoY5Genb7zx5luyHP/s9oc3PtRYy/ZLpD/ArwcFgDzBxsBjQIIWCGAnxZZRMApGwSgYAQAApJ9C//wzAS4AAAAASUVORK5CYII=","orcid":"","institution":"Benson Idahosa University","correspondingAuthor":true,"prefix":"","firstName":"Ikenna","middleName":"C.","lastName":"Maduako","suffix":""},{"id":459229343,"identity":"baa1d666-2f2a-480d-8056-c0a7b56d4b12","order_by":1,"name":"Chidinma N. Igwe","email":"","orcid":"","institution":"Imo State University Owerri","correspondingAuthor":false,"prefix":"","firstName":"Chidinma","middleName":"N.","lastName":"Igwe","suffix":""},{"id":459229344,"identity":"0903a89e-f453-4476-87b1-757b9991836a","order_by":2,"name":"Chinweokwu V. Ego","email":"","orcid":"","institution":"University of Benin","correspondingAuthor":false,"prefix":"","firstName":"Chinweokwu","middleName":"V.","lastName":"Ego","suffix":""},{"id":459229345,"identity":"d69b0321-ef77-442e-a503-d52e1b355671","order_by":3,"name":"Osarhieme T. Okugbo","email":"","orcid":"","institution":"Benson Idahosa University","correspondingAuthor":false,"prefix":"","firstName":"Osarhieme","middleName":"T.","lastName":"Okugbo","suffix":""},{"id":459229346,"identity":"c421a45c-b431-431a-a761-1b5112a3735c","order_by":4,"name":"Uzoamaka N. Ngwoke","email":"","orcid":"","institution":"Benson Idahosa University","correspondingAuthor":false,"prefix":"","firstName":"Uzoamaka","middleName":"N.","lastName":"Ngwoke","suffix":""},{"id":459229347,"identity":"2ee628bd-0198-436a-82a3-504074d61597","order_by":5,"name":"Abraham O. Ekhegbesela","email":"","orcid":"","institution":"Benson Idahosa University","correspondingAuthor":false,"prefix":"","firstName":"Abraham","middleName":"O.","lastName":"Ekhegbesela","suffix":""}],"badges":[],"createdAt":"2025-05-20 09:28:42","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-6706130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6706130/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83203029,"identity":"88d81c12-5a29-4adb-8145-4f7aa6eff71c","added_by":"auto","created_at":"2025-05-21 06:52:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1408050,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6706130/v1/3297648ba90b9ee794cde1bc.png"},{"id":83204280,"identity":"ec97b779-ee19-46e0-9d88-b21d7e0d0f84","added_by":"auto","created_at":"2025-05-21 07:08:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":312849,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6706130/v1/1e751420a28d85506ea17f22.png"},{"id":83203032,"identity":"a18b58ee-dce4-452b-b773-9e5010ec7f54","added_by":"auto","created_at":"2025-05-21 06:52:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":267461,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6706130/v1/c83f13fcaee9b8665fa36af7.png"},{"id":83203038,"identity":"69b461b0-6967-4ce6-a1e3-1cd600f9d383","added_by":"auto","created_at":"2025-05-21 06:52:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20524653,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6706130/v1/377e3681772aa27fd94a099c.png"},{"id":83206212,"identity":"0eba4526-5d65-4bc3-8904-6ff99686e8a7","added_by":"auto","created_at":"2025-05-21 07:24:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":30547516,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6706130/v1/35ae7e16-c0b8-4314-b1a2-7650f4b85485.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eHesperetin and naringenin relieve neurotoxicity and histological distortions in benzo[a]pyrene-challenged rats\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCivilization's progress is intertwined with industrialization and urbanization, leading to increased reliance on fossil fuels for energy production. The combustion of these fuels, such as coal and oil, generates polycyclic aromatic hydrocarbons (PAHs) as byproducts. These environmental contaminants, including benzo[a]pyrene (BaP), are widespread due to extensive industrial activities [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. PAHs can persist in the environment, adhering to particles of various sizes and accumulating in organisms. While individual PAHs may not be definitively classified as human carcinogens, PAH mixtures are recognized as cancer-causing agents, particularly in occupations with high exposure, such as coal mining and soot-related industries [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. BaP, a classical compound for investigating the impact of this type of PAH.\u003c/p\u003e \u003cp\u003e Currently, neurological disorders are on the rise. In the US for example, an estimated 1.1\u0026nbsp;million adults develop brain disorders annually, with over 13\u0026nbsp;million individuals living with these conditions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Also, neurological disorders are becoming more common in Sub-Saharan Africa and this increase is linked to factors such as rising vehicular traffic, alcohol and substance abuse [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The economic burden of mental illness is substantial, with an estimated \u0026euro;810\u0026nbsp;billion spent in Europe in 2010 and a global cost of \u003cspan\u003e$\u003c/span\u003e604\u0026nbsp;billion for dementia in the same year [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These figures underscore the critical need for research and management strategies to address this growing health concern. The brain, with its high metabolic activity, demands a constant supply of oxygen and glucose. This metabolic intensity makes it vulnerable to oxidative stress, leading to lipid peroxidation (due to its rich lipid content), disruption of signaling pathways, and dysregulation of gene expression [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The brain's limited regenerative capacity and moderate antioxidant defenses further exacerbate its susceptibility to reactive oxygen species (ROS)-mediated damage [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOxidative stress has been implicated in the pathogenesis of various neurological disorders, including Parkinson's disease, Alzheimer's disease, and schizophrenia, through mechanisms involving excitotoxicity, apoptosis, and cellular signaling alterations [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. While DNA adduct formation is a well-established mechanism of PAH carcinogenicity, oxidative stress playing a crucial role. PAHs can induce redox cycling, leading to ROS generation and overwhelming endogenous antioxidant systems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The lipophilic nature of PAHs enables their penetration of the blood-brain barrier, facilitating their entry into brain tissue and exerting their deleterious effects [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntioxidant compounds offer a promising avenue for mitigating oxidative stress. These compounds can scavenge free radicals, enhance antioxidant enzyme activity, chelate metal ions, and boost endogenous antioxidant levels [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Flavonoids, a class of polyphenolic phytochemicals, exhibit potent antioxidant properties due to their numerous hydroxyl and methoxy groups [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, flavonoids demonstrate regulatory effects on various cellular processes, including inflammation and carcinogenesis, by modulating gene expression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This evidence has spurred the investigation of compounds like curcumin, resveratrol, and lycopene in clinical trials for various cancers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHesperetin (5,7,3\u0026prime;-trihydroxy-4\u0026prime;-methoxyflavanone), a flavanone abundant in citrus fruits, particularly oranges, is obtained from hesperidin through deglycosylation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Studies have demonstrated hesperetin's potential in inhibiting cell proliferation, promoting apoptosis, and suppressing angiogenesis in rat colon carcinogenesis models [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In human colon adenocarcinoma cells (HT-29), hesperetin has been shown to inhibit cell growth, downregulate tumor markers, and upregulate Notch1 mRNA and protein levels [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While Naringenin, chemically known as 5,7-dihydroxy-2-(4-hydroxyphenyl) chroman-4-one, an flavonone primarily found in grapefruit [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], exhibits various biological activities. These include adipogenesis inhibition, reduced insulin sensitivity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], suppression of atopic dermatitis in mice [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], protection against carbon tetrachloride-induced nephrotoxicity in mice [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and amelioration of hyperglycemia-mediated inflammation in experimental diabetic rats [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis research examined how oxidative stress contributes to the neurotoxic impact of BaP on rat brain tissue and evaluated the neuroprotective effects of Hesperetin and Naringenin against oxidative injury caused by BaP.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and reagents\u003c/h2\u003e \u003cp\u003eBenzo[a]pyrene (\u0026gt;\u0026thinsp;95%) was acquired from Sigma-Aldrich, Missouri, USA. Hesperetin (98%) and Naringenin (98%) were procured from AK Scientific, Union City, California, USA. All other chemicals utilized in this research were of analytical grade, obtained from Sigma-Aldrich, located in St. Louis, Missouri, USA, and British Drug Houses in Poole, Dorset, UK.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animals\u003c/h2\u003e \u003cp\u003eThirty-two adult Wistar strain male albino rodents (180\u0026ndash;200g) were procured from the Animal House, Department of Medical Biochemistry. The animals were housed in ventilated cages under controlled temperature conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and a standardized 12-hour light/dark cycle. The animals were provided with unrestricted availability of feed and drinking water. After adapting to their environment for 7 days, the animals were arbitrarily assigned to one of four separate cohorts, each comprising seven animals. All experimental protocols were carried out following the guidelines of the Benson Idahosa University Ethics Committee Animal Handling Protocol (ECBIUAHP-0012/med/089).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Study design\u003c/h2\u003e \u003cp\u003eThe animals were grouped and treated as below:\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCTRL\u003c/strong\u003e \u003cp\u003eCORN OIL (2.5 ml/kg)\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBaP\u003c/strong\u003e \u003cp\u003eBENZO[A]PYRENE (4 mg/kg)\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBaP\u0026thinsp;+\u0026thinsp;NAR\u003c/strong\u003e \u003cp\u003eBENZO[A]PYRENE (4 mg/kg)\u0026thinsp;+\u0026thinsp;NARINGENIN (50 mg/kg)\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBaP\u0026thinsp;+\u0026thinsp;HPN\u003c/strong\u003e \u003cp\u003eBENZO[A]PYRENE (4 mg/kg)\u0026thinsp;+\u0026thinsp;HESPERETIN (50 mg/kg)\u003c/p\u003e \u003c/p\u003e \u003cp\u003eTreatment was performed via oral gavage daily for 28 consecutive days. The dose of BaP, Hesperetin and Naringenin were selected based on previous studies [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Benzo[a]pyrene was slightly solubilized in dimethylsulfoxide and mixed in corn oil, while Naringenin and Hesperetin were dissolved in corn oil.\u003c/p\u003e \u003cp\u003eTwenty-four hours following the final treatment, the subjects were euthanized using the cervical dislocation method. Brains were promptly removed and rinsed in chilled potassium chloride buffer. For biochemical analyses, brain tissues were weighed, blended in chilled phosphate-buffered solution (pH 7.4) at a 1:4 weight-to-volume ratio using a glass homogenizer. Centrifugation of the homogenates was performed at 10,000 g for 15 minutes at 4\u0026deg;C. The resulting supernatants, following the mitochondrial phase, were kept at -80\u0026deg;C until further investigation. For microscopic tissue assessment, neural samples were preserved in 10% formaldehyde for subsequent processing and embedding.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Biochemical analyses\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Assessment of neuro-anti-oxidative biomarkers\u003c/h2\u003e \u003cp\u003eCatalase activity in the brain was measured by monitoring the decrease in absorbance at 240 nm as hydrogen peroxide is consumed, according to the method described by Claiborne [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Hydrogen peroxide concentration, which correlates with absorbance at this wavelength, was quantified by applying an extinction coefficient value of 0.0436 mM⁻\u0026sup1;cm⁻\u0026sup1;. SOD activity was evaluated using the procedure described by Misra and Fridovich [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], which measures the inhibition involving adrenaline autoxidation to its oxidation product at pH 10.2. Lipid peroxidation was quantified using the Varshney and Kale method, modified by Farombi [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. TBARS (thiobarbituric acid reactive compounds) and MDA (malondialdehyde) were determined, with MDA forming a pink complex with 2-thiobarbituric acid under acidic conditions, detectable at 532 nm.\u003c/p\u003e \u003cp\u003eGST (Glutathione S-transferase) activity was assessed following the procedure outlined by Habig [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The enzyme function was evaluated through the coupling of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione. This shift in the absorbance spectrum to 340 nm allows for direct measurement of enzyme activity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], while the concentration of reduced glutathione (GSH) was measured using the procedure described by Beutler [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Glutathione levels were measured using Ellman's reagent 5,5\u0026prime;-dithiobis (2-nitrobenzoic acid) (DTNB). DTNB reacts with sulfhydryl groups, producing a yellow product with maximum absorbance at 412 nm [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Xanthine oxidase (XO) activity was assessed using the method of Bergmeyer [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The total sulfhydryl group (-TSH) in biological sample was estimated according to Ellmans [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. It relies Ellmans reagent reaction with sulfhydryl groups to produce a yellow coloured complex.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeuro assessment of pro-inflammatory markers\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMyeloperoxidase (MPO) activity was determined using a previously described method [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e35\u003c/span\u003e] Levels of nitric oxide (NO) were determined by assessing nitrite concentrations using a Griess reagent according to a standard protocol [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) were quantified in the brain using commercial ELISA kits from Elabscience (China) and analyzed on a SpectraMax microplate reader from Molecular Devices (USA) following their guidelines.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMonitoring of reactive oxygen and nitrogen species (RONS) production in brain tissue\u003c/b\u003e \u003c/p\u003e \u003cp\u003eReactive oxygen and nitrogen intermediates (RONS) production was assessed within brain tissues using a previously described method [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This relies on oxidising 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) into 2',7'-dichlorofluorescein (DCF) by tissue-derived RONS. Succinctly, 10 \u0026micro;L of the tissue homogenate was mixed with 5 \u0026micro;L of DCFH-DA (200 \u0026micro;M; achieving a final concentration of 5 \u0026micro;M) in 150 \u0026micro;L of 0.1M potassium phosphate buffer (pH 7.4) along with 35 \u0026micro;L of distilled water. After DCFH-DA oxidation, the fluorescence signal of DCF was detected at excitation and emission wavelengths of 488 nm and 525 nm, respectively, using a SpectraMax microplate reader. The DCF generation rate was a percentage relative to the control group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExamination of Tissue Morphology\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBrain samples were preserved in 10% formalin for 7 days. Following fixation, tissues were processed for paraffin embedding. This involved serial dehydration in graded ethanol solutions, followed by clearing in xylene. Tissues embedded in paraffin were sliced into 5 \u0026micro;m thick sections with a microtome. The tissue sections were placed on glass slides, cleared of paraffin using xylene, and gradually rehydrated through a series of graded ethanol solutions. Afterward, the sections were stained with hematoxylin and eosin (H\u0026amp;E) and observed under a light microscope. Microscopic tissue evaluation was carried out following the procedures described by Hotchkis and McManus [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe data were subjected to one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test for multiple comparisons, utilizing GraphPad PRISM 8 software. A p-value of less than 0.05 was considered statistically significant. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003eNAR\u0026thinsp;+\u0026thinsp;HPN improve neurotoxicity in BaP-challenged rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan\u003e1\u003c/span\u003eA \u003cstrong\u003e\u0026amp; B)\u003c/strong\u003e, Antioxidant enzyme and glutathione levels: BaP exposure caused a marked (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decline in Glutathione S-transferase (GST) activities, as well as in the concentration of reduced Glutathione (GSH) compared to the control group. Combined treatment with NAR\u0026thinsp;+\u0026thinsp;HPN dosed at 50 mg/kg each reversed the BaP-induced decreases, restoring GST, and GSH levels. (Fig. \u003cspan\u003e1\u003c/span\u003eC, D \u003cstrong\u003e\u0026amp; E)\u003c/strong\u003e: antioxidant enzyme activities and total sulfhydryl group (TSH) levels: Similarly, BaP exposure led to a marked (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduction in CAT and SOD activities, as well as a reduction in TSH levels, compared to the control group. NAR\u0026thinsp;+\u0026thinsp;HPN co-administration significantly attenuated these BaP-induced reductions in CAT, SOD, and TSH. Figure \u003cspan\u003e1\u003c/span\u003eF, G \u003cstrong\u003e\u0026amp; H)\u003c/strong\u003e: oxidative stress markers and xanthine oxidase (XO) activity: BaP administration markedly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) elevated the concentrations of lipid peroxidation (LPO), reactive oxygen and nitrogen intermediates, as well as XO activity in rat brain, relative to the control group. Co-administration of NAR\u0026thinsp;+\u0026thinsp;HPN significantly reduced (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) these BaP-induced elevations in LPO, RONS, and XO activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNAR\u0026thinsp;+\u0026thinsp;HPN diminishes BaP-mediated increase in inflammatory indices in the brain of rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NAR\u0026thinsp;+\u0026thinsp;HPN effect against BaP-instigated increase in the indices of inflammation is presented in Fig. \u003cspan\u003e2\u003c/span\u003e. BaP treatment alone increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) NO level and MPO activity in rats brain relative to control. These mediated increases were reduced (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when the rats were administered with NAR\u0026thinsp;+\u0026thinsp;HPN. Figure \u003cspan\u003e3\u003c/span\u003e: Exposure to BaP led to a marked increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in levels of the proinflammatory mediators TNF-\u0026alpha; and IL-1\u0026beta; within the brains of rats relative to the control group. Such increases were notably reduced following NAR\u0026thinsp;+\u0026thinsp;HPN administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNAR\u0026thinsp;+\u0026thinsp;HPN abrogated histological alterations in rats brain following BaP challenge\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe photomicrographs of brain from rats exposed to BaP and NAR\u0026thinsp;+\u0026thinsp;HPN were examined microscopically in (Fig. \u003cspan\u003e4\u003c/span\u003eA, B \u003cstrong\u003e\u0026amp; C).\u003c/strong\u003e The images observed from the control and NAR\u0026thinsp;+\u0026thinsp;HPN-treated rats were consistent with intact brain morphological and histological architecture, while the group challenged with BaP presented altered histological architecture.\u003c/p\u003e"},{"header":"4. Discussions","content":"\u003cp\u003eThis investigation aimed to evaluate the impact of Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), on brain antioxidant capacity and structural integrity. A 28-day BaP administration protocol at 2 mg/kg, established by Maciel et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], was employed to mimic exposure levels observed in individuals with high consumption of grilled foods. The dosing regimen complied with the standards set by the Organization for Economic Co-operation and Development (OECD) for repeated-dose toxicity assessments. Other studies have utilized higher BaP doses (25\u0026ndash;200 mg/kg) in single-dose exposure models to mimic specific scenarios like occupational exposures [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Approximately 40% of orally administered BaP is bioavailable, resulting in a relatively low effective concentration at target tissues [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Furthermore, we hypothesized that an intervention strategy involving natural products such as Naringenin (NAR) and Hesperetin (HPN) could mitigate BaP-induced neurotoxicity.\u003c/p\u003e \u003cp\u003eThe toxic effect of BaP is linked to its highly reactive intermediate and the production of reactive oxygen species (ROS) during its bioactivation and can induce oxidative stress, initiate DNA damage and disrupt cellular activities [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. We investigate strategies to mitigate BaP neurotoxicity, which is attributed to compromised antioxidant defenses, oxido-inflammatory responses and the formation of reactive oxygen species [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our approach involves the development of preventive strategies, including the use of plant-derived natural products to counteract BaP-induced neurotoxicity and promote neuronal health [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. SOD and CAT represent key antioxidant enzymes that act cooperatively to neutralize RONs. SOD facilitates the conversion of superoxide ions (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e) to hydrogen peroxide, thereby reducing oxidative stress. Subsequently, CAT decomposes H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into water and oxygen, completing the detoxification process and maintaining cellular redox balance. The administration of BaP to rats in the study markedly diminished SOD and CAT activities, indicating enzyme inhibition, thus accumulation of toxic superoxide radicals and hydrogen peroxide molecules in the brain, causing oxidative stress. However, co-administration of NAR and HPN to rats challenged with BaP, significantly enhanced SOD and CAT activities, suggesting NAR and HPN antioxidant capacity, which may mitigate BaP induced neurotoxicity.\u003c/p\u003e \u003cp\u003eOxidative stress occurs when the delicate balance between antioxidants and pro-oxidants is disrupted, favouring the dominance of pro-oxidants. This imbalance allows ROS to accumulate, causing cellular damage [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Free radicals generated from oxidative stress can undermine both enzymatic and non-enzymatic cellular antioxidant defenses, leading to macromolecular damage, enhanced lipid peroxidation, and increased reactive oxygen and nitrogen species levels [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Elevated RONS production plays a significant role in triggering and advancing injuries as well as many systemic disorders [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Elevated levels of RONS, LPO, and XO activity mark the characteristic profile of oxidative stress. Conversely, this imbalance is often accompanied by decreased activity of the enzymatic antioxidant GST and diminished concentration of GSH and total thiol TSH, indicating compromised cellular antioxidant defenses. Our study reveals that exposure to BaP alone led to significant decline in brain GSH and TSH levels, accompanied by increases in LPO, RONS levels, and xanthine oxidase activity. These findings suggest that BaP-induced oxidative stress can cause neuronal toxicity, which is consistent with [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, co-administration of NAR and HPN significantly enhanced GST activity and restored GSH and TSH levels, indicating their potential neuroprotective impact in response to BaP-stimulated oxidative stress. Similarly, the combined administration of NAR and HPN notably lowered LPO, RONS, and XO activity in the treated rats, demonstrating antioxidant characteristics. The observed outcomes associated with NAR and HPN are likely due to their inherent antioxidant properties [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurther, nitric oxide is capable of interacting with superoxide anion (O\u003csub\u003e2\u003c/sub\u003e-), leading to the generation of peroxynitrite (ONOO-), a deleterious RNS, while MPO, a heme-containing enzyme primarily released by neutrophils is closely linked to the development of oxidative stress and inflammation. However, unabated degranulation exacerbates inflammation, and tissue damage ensues [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The proinflammatory cytokines, IL-1β and TNF-α, are star players during inflammatory processes, facilitating cell recruitment to injury sites [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Elevated concentrations of IL-1β and TNF-α in BaP-exposed rats brains indicate a pronounced inflammatory response. Increased IL-1β and TNF-α can, in turn, activate iNOS leading to overproduction of NO perpetuate oxidative stress and tissue anomaly [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our findings are consistent with previously reported data on BaP-induced changes in inflammation bioamarkers [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The elevated levels of neuronal NO, IL-1β and TNF-α, as well as increased MPO activity, observed in BaP-teated rats, suggest that BaP exposure may lead to neuronal dysfunction via inflammatory and nitrosative stress. In contrast, treatment with NAR and HPN mediated significant reductions in NO, IL-1β, TNF-α levels, and MPO activity in BaP-challenged rats, demonstrating the anti-inflammatory effects of NAR and HPN against BaP-induced neuroinflammatory responses.\u003c/p\u003e \u003cp\u003eThe histopathological assessment provides a comprehensive evaluation of tissue architecture and disease progression, visually confirming the biochemical alterations observed in the study. Our findings revealed that administration of BaP alone caused pronounced degeneration of cortical neurons and Purkinje neurons (PNCA3) in treated rats. Furthermore, histomorphometric analysis demonstrated significant reductions in the densities of cortical neurons, PNCA3, granule cell layer of the dentate gyrus (GCDG), and white matter of the posterior cerebral cortex (WDPC) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. These histopathological and histomorphometric alterations observed in the brains of rats treated with BaP are likely associated with oxidative damage and neuronal inflammation. Our observations align with previous study of Farombi et al [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Cotreatments with NAR and HPN as we observed, reversed these histological and morphometrical changes, preserving brain structures and neurons in a manner comparable to the control. This finding aligns with biochemical effects of NAR and HPN against BaP-induced neurotoxicity in rats.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe present data suggest that NAR and HPN effectively mitigated neuronal toxicity in BaP-treated mice via multiple biochemical mechanisms. They also protected against BaP-induced inflammation and supported antioxidant defenses. The study's observed health benefits of NAR and HPN highlight their potential as therapeutic phytochemicals for addressing BaP-induced brain toxicity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received no funding from either public, private or commercial bodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIkenna C. Maduako\u003c/strong\u003e: Conceptualisation, Methodology, Data curation, writing- original draft. \u003cstrong\u003eChidinma N. Igwe\u003c/strong\u003e: writing original draft. \u003cstrong\u003eChinweokwu V. Ego:\u003c/strong\u003e Project administration and proofreading the original draft. \u003cstrong\u003eUzoamaka N. Ngwoke:\u003c/strong\u003e Proofreading the original draft. \u003cstrong\u003eAbraham O. Ekhegbesela\u003c/strong\u003e: data curation. \u003cstrong\u003eEsther A. Omoyajowo\u003c/strong\u003e: data curation, writing original draft\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts to declare\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Dr. Iramofu Dominic of Georgia Institute of Technology, USA, for the gift of Benzo[a]pyrene used in this study and the University of Buea, Cameroon.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIARC Monographs on the Evaluation of Carcinogenic Risks to Humans (2012) Vol. 100F: Some Chemicals Present in Industrial and Consumer Products, Poisons and Pesticides\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eATSDR (Agency for Toxic Substances and Disease Registry) (2010) Toxicological Profile for Polycyclic Aromatic Hydrocarbons (PAHs)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIgwe CN, Ngwoke UN, Okugbo OT, Ikpowonsa-Eweka O, Ego CV, Omoyajowo EA, Maduako IC (2025) Air Quality and Public Health in Metropolitan Cities in Nigeria: Implications. 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Afr J Med Med Sci 36:103\u0026ndash;108\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarombi EO, Awogbindin IO, Owoeye O, Maduako IC, Ajeleti AO, Owumi SE (2020) Kolaviron via anti-inflammatory and redox regulatory mechanisms abates multi-walled carbon nanotubes-induced neurobehavioral deficits in rats. Psychopharmacology. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00213-019-05432-8\u003c/span\u003e\u003cspan address=\"10.1007/s00213-019-05432-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Benson Idahosa University","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":"Benzo[a]pyrene, Hesperetin, Naringenin, Neurotoxicity, chemoprevention","lastPublishedDoi":"10.21203/rs.3.rs-6706130/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6706130/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e \u003cp\u003eEnvironmental pollutants like benzo[a]pyrene (BaP), which belongs to the PAH family, generated from fossil fuel combustion, pose a neurotoxic risk. Their lipophilic nature allows them to breach the blood-brain barrier and trigger oxidative stress. This research explored the neuroprotective effectiveness of the flavonoids hesperetin together with naringenin against BaP-induced neurotoxicity in a rodent model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 28 male Wistar rats were randomly distributed into four experimental groups and treated with BaP alone or in combination with hesperetin or naringenin for 14 days.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results demonstrated that BaP exposure led to significant depletion of glutathione, inhibition of glutathione peroxidase, and elevation of lipid peroxidation, hydrogen peroxide, and xanthine oxidase activity in rat brain tissue. Histopathological analysis revealed neuronal damage characterized by pyknotic cells in the cerebrum and shrunken Purkinje cells in the cerebellum. Co-administration of hesperetin markedly attenuated these oxidative stress markers and prevented histopathological alterations. Naringenin also exhibited some protective effects against structural damage.\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e \u003cp\u003eOur findings suggest that hesperetin and naringenin can mitigate BaP-induced neurotoxicity by bolstering antioxidant defenses and preserving brain tissue integrity. These results highlight the potential neuroprotective benefits of flavonoid-rich citrus fruits in combating the adverse effects of environmental pollutants like BaP.\u003c/p\u003e","manuscriptTitle":"Hesperetin and naringenin relieve neurotoxicity and histological distortions in benzo[a]pyrene-challenged rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-21 06:52:13","doi":"10.21203/rs.3.rs-6706130/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":"b7a25fc1-13d4-4772-8adf-003e53245af8","owner":[],"postedDate":"May 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48783071,"name":"Applied Biochemistry"}],"tags":[],"updatedAt":"2025-05-21T06:52:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-21 06:52:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6706130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6706130","identity":"rs-6706130","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}