Lutein and Zeaxanthin Provide Protection Against Aflatoxin B1-Induced Liver and Kidney Toxicity: Insights from in Silico and in Vivo Studies

Lutein and Zeaxanthin Provide Protection Against Aflatoxin B1-Induced Liver and Kidney Toxicity: Insights from in Silico and in Vivo Studies | 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 Lutein and Zeaxanthin Provide Protection Against Aflatoxin B1-Induced Liver and Kidney Toxicity: Insights from in Silico and in Vivo Studies Solomon Owumi, Joseph Chimezie, James A. Owolabi, Jesse C. Nwaokolo, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8340180/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Exposure to aflatoxin B 1 (AFB 1 ) poses significant threats to food safety, increases food insecurity, and endangers public health due to its pronounced organ toxicity and carcinogenicity, especially affecting the hepatorenal system. This study integrates network pharmacology and molecular docking analyses to investigate the protective roles of the natural carotenoids lutein (LUT) and zeaxanthin (ZEA) against AFB 1 -induced toxicity. Key hub genes-ALB, IL2, LGALS3, SRC, REN, EGFR, PRKACA, LCN2, F2, and TTR- implicated in carcinogenesis, stress response, cellular homeostasis, and tissue remodelling were identified via Network Pharmacology analysis. Molecular docking demonstrated that LUT (-9.0 kcal/mol) and ZEA (-9.5 kcal/mol) have higher affinity to inflammation-related proteins (LGALS3) than AFB1 (-8.4 kcal/mol), suggesting their strong anti-fibrotic and antioxidant potentials. Lutein also showed the highest binding affinity for REN (-9.5 kcal/mol) compared to AFB 1 (-8.8 kcal/mol), suggesting a specific mechanism through which LUT may influence the RAAS pathway to reduce AFB 1 -induced kidney damage. In vivo experiments using Wistar rats (N = 25; 10 weeks old, weighing 220 ± 20 g) randomly assigned to five groups and treated as follows: Control group (corn oil only, 2 mL/kg), AFB1 only (75 µg/kg), LUT/ZEA only (100 mg/kg), AFB1 (75 µg/kg) plus LUT/ZEA1 (100 mg/kg), and LUT/ZEA2 (200 mg/kg) per os for 28 days. And followed by the assessments of liver and kidney function, serum lipid profiles, enzymatic and non-enzymatic antioxidant levels, oxidative stress markers, inflammatory mediators, DNA damage, and apoptosis biomarkers were conducted using spectrophotometric methods, confirming that LUT/ZEA administration alleviated AFB 1 -induced liver and kidney damage, reduced oxidative stress, inflammation, DNA damage, and apoptosis in a dose-dependent manner. Histopathological analyses further validated these protective effects. Overall, the study highlights the potential of LUT and ZEA as natural agents for mitigating AFB 1 toxicity, offering promising strategies to improve food safety and reduce health risks associated with mycotoxin exposure. Network pharmacology Aflatoxin B1 hepatorenal toxicity lutein and zeaxanthin oxido-inflammatory and DNA damage apoptosis and molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Introduction Aflatoxin B 1 (AFB 1 ) stands as the most potent aflatoxin produced by select Aspergillus species and poses a significant threat to food safety worldwide. Its frequent presence in crops such as maize and peanuts, especially in warm and humid regions [ 1 – 3 ], has led regulatory agencies—including those in the European Union and United States—to establish safety limits for human intake ranging from 2 to 30 µg/kg [ 4 , 5 ]. Nevertheless, despite regulatory efforts and multiple mitigation strategies, AFB 1 contamination remains persistent in food and feed, contributing to outbreaks of aflatoxicosis and a host of related diseases, including liver cancer, severe hepatitis, Reye's syndrome, gallbladder cancer, kwashiorkor, and respiratory illnesses [ 5 – 9 ]. The toxicity of AFB 1 is strikingly broad, affecting organs such as the liver, kidneys, pancreas, brain, heart, and gonads in both humans and animals [ 3 ]. The liver is particularly susceptible, serving as the primary site for AFB 1 metabolism via the cytochrome P450 enzyme system—most notably CYP1A2 and CYP3A4. This process generates a highly reactive epoxide (AFB 1 -8,9-epoxide), which forms DNA adducts like AFB 1 -N7-guanine, resulting in genetic mutations and carcinogenesis [ 10 , 11 ]. Mechanistic studies have revealed that AFB 1 induces toxicity through several interrelated pathways: oxidative stress, inflammation, and apoptosis [ 10 – 15 ]. Specifically, AFB 1 suppresses antioxidant defenses, stimulates the production of reactive oxygen species (ROS) and lipid peroxidation (by inhibiting the Nrf2/HO-1 pathway) [ 12 , 13 ], and promotes mitochondrial-mediated apoptosis by modulating apoptotic markers such as cytochrome c, caspase 3, caspase 8, and Bcl-2-associated X protein (Bax), while downregulating B-cell lymphoma 2 (Bcl-2) [ 13 – 15 ]. Lutein (LUT) and zeaxanthin (ZEA), two naturally occurring xanthophyll carotenoids found abundantly in green leafy vegetables, corn, and egg yolks, have garnered attention for their health-promoting properties that extend beyond ocular benefits[ 16 ]. Recent research underscores their antioxidant, anti-inflammatory, and anti-apoptotic activities in models of neurotoxicity, hepatotoxicity, and nephrotoxicity [ 17 – 23 ]. LUT and ZEA have been shown to reduce oxidative stress by scavenging ROS, preserve cellular membrane integrity, suppress pro-inflammatory cytokines (e.g., TNF-α, IL-6) via nuclear factor kappa-light-chain-enhancer of activated B cells NF-κB inhibition [ 24 – 26 ], and modulate key molecular pathways including peroxisome proliferator-activated receptor gamma (PPARγ) and nuclear factor erythroid 2-related factor 2 (Nrf2). These effects help restore cellular homeostasis and limit tissue injury [ 27 , 28 ]. Despite mounting evidence of the protective effects of LUT and ZEA, their role in counteracting AFB1-induced hepatorenal toxicity remains insufficiently explored. This research hypothesizes that LUT and ZEA can attenuate AFB 1 -driven oxidative stress, inflammation, and apoptosis in hepatic and renal tissues. To test this, the study integrates molecular docking, network pharmacology, and in vivo analysis to investigate the mechanisms and potential target proteins involved in the protective actions of these carotenoids against AFB 1 toxicity. The insights gained from this work may guide the development of dietary strategies to mitigate the health risks posed by AFB 1 exposure, particularly in populations at elevated risk due to persistent contamination. In revealing the molecular underpinnings of LUT and ZEA’s protective effects, this study seeks to contribute meaningfully to the advancement of nutritional interventions and public health safeguards against aflatoxicosis. Materials and Methods Network Pharmacology The 3D structure of Aflatoxin B 1 , Lutein, and Zeaxanthin were obtained through the PubChem database ( https://pubchem.ncbi.nlm.nih.gov/ ). Potential targets of AFB 1 , Lutein and Zeaxanthin were predicted using the PharmMapper ( https://www.lilabecust.cn/pharmmapper/ ) and SwissTargetPrediction ( https://www.swisstargetprediction.ch/ ) databases assessed on 15th October, 2025. The SDF file of AFB 1 , Lutein and Zeaxanthin was uploaded to the PharmMapper, the dataset of human protein targets only (v2010, 2241) in PharmMapper, the default values for the rest parameters were kept, the job was submitted, and the screening results were awaited. Simultaneously, the SMILES from PubMed were uploaded to the SwissTargetPrediction database, and the results were saved as a CSV file. Subsequently, the targets collected from the PharmMapper and SwissTargetPrediction databases were filtered, and duplicates were removed using Microsoft Excel 2025 for subsequent analysis. The potential targets for hepatorenal toxicity were discovered using GeneCards ( https://www.genecards.org/ ) assessed on 15th October, 2025, using “hepatorenal toxicity” as the keyword to recruit the targets of hepatorenal toxicity. Predicted results were exported, and the targets collected from the GeneCards database were filtered accordingly for subsequent analysis. Intersection targets of AFB 1 , LUT/ZEA, and Hepatorenal toxicity were obtained using the Venny 2.1 website. The PPI (protein–protein interaction) network was collected by STRING 12.0 ( https://string-db.org/ ), proteins were obtained on the STRING 12.0 database accessed on 17th October, 2025, and this given list of interactions was imported to predict the interaction of identified differentially expressed proteins (DEPs). The file of DEPs obtained from STRING was imported into Cytoscape (version 3.10.3) to construct the PPI network. The CytoHubba app plug-in in Cytoscape was used to compute the Maximum Clique Centrality (MCC) for identifying hub genes, thus creating a PPI network of the hub genes. Shiny G.O (0.85) accessed on 17th October, 2025, was used to perform gene ontology, a gene functional classification system that provides dynamically updated terms to comprehensively describe the properties and products of targets and the enrichment analysis of intersection targets was performed The Kyoto Encyclopedia of Genes and Genomes (KEGG) can systematically analyze the gene functions and metabolic terms pathways involved in the target gene functions and metabolic pathways involved in the target genes, which is helpful to fully understand the overall effect of the predicted targets in the organism. Molecular Docking Molecular docking was performed to assess the binding affinity and potential interaction sites between aflatoxin B1, zeaxanthin, lutein, and the selected hub genes, to elucidate the possible inhibitory mechanisms. The 3D protein data bank (PDB) files of key targets (ALB, IL2, LGALS3, SRC, REN, EGFR, PRKACA, LCN2, F2, TTR) were retrieved from the RCSB database ( https://www.rcsb.org/ ), assessed on 17th October, 2025. Molecular docking analysis using PyRx was conducted to predict the molecular interactions between all the genes, Aflatoxin B 1, Lutein, and Zeaxanthin. Ligand preparation, protein preparation, binding sites recognition, docking, and visualization of docking modes were done using Discovery Studio 2021 software. Chemical Lutein & Zeaxanthin were purchased from Costco Wholesale Corporation, USA. Aflatoxin B 1 powder (AFB 1 , purity ≥ 98.0%, #1162-65-8, C 17 H 12 O 6 , MW: 312.27g), high-density lipoprotein (HDL; Cat. No. MAK331), total cholesterol (TC; Cat. No. CS0005), and triglycerides (TG; Cat. No. MAK266) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Aspartate aminotransferase (AST; Cat. No. AS101), Alanine aminotransferase (ALT; Cat. No. AL146), creatinine (Cat. No. CR510), and urea (Cat. No. UR1068) assay kits were purchased from Randox Laboratories Limited (Crumlin, UK). Enzyme-linked Immunosorbent Assay kits for estimation of p53 (E-EL-H0910), Bcl-2 (E-EL-R0096), and BAX (E-EL-R0098) were purchased from E-labscience Biotechnology Co., Ltd, Wuhan, China. Monosodium phosphate (CAS No.: 7558–80 − 7), disodium hydrogen phosphate (CAS No.: 7558–79 − 4), sodium carbonate (CAS No.: 497–19 − 8), sodium hydroxide (CAS No.: 1310–73 − 2), sodium–potassium tartrate (CAS No.: 6381–59 − 5), and sodium chloride (CAS No.: 7647–14 − 5) were obtained from BDH Ltd. (Poole, Dorset, UK) and William Hopkins Ltd. (Birmingham, UK). All other biochemical reagents and chemicals were obtained commercially and of analytical grade. Animal care and welfare Ten (10) weeks-old male Wistar rats (180–230 g) were obtained from the Faculty of Veterinary Medicine, University of Ibadan College of Medicine (Ibadan, Nigeria). The experimental rats were acclimatised for 7 days before the commencement of the experimental period and housed in plastic cages with unrestricted access to standard feed (Breedwell® Feeds Limited, Ibadan, Nigeria) and freshwater. All rats were housed under standard laboratory conditions, with a natural photoperiod featuring a daily cycle of light and dark lasting approximately 12 hours at 23 ± 2°C. This experiment adheres to the 3Rs (replacement, reduction, and refining) guidelines for the use and care of experimental animals, approved by the University of Ibadan Animal Care and Use Research Ethics Committee (ACUREC No. UI-ACUREC/068–0524/06) and conforms with the NIH publications volume 25, No.28 and revised in 1996 and EU Directive 2010/63/EU guidelines. Additionally, the animals' health was closely monitored, and appropriate measures were taken daily to ensure their maximum welfare, including checking fur condition, animal mobility, and body weight. Animals showing weight loss exceeding an acceptable limit, significant trauma, and inactivity for more than 24 hours were culled from the study. Experimental design and treatment Twenty-five (25) male Wistar rats were employed for the current study and were randomly assigned into five (5) cohorts of eight animals as follows: The control cohort received corn oil 2 mL/kg per os Aflatoxin B 1 cohort ( AFB 1 ) received aflatoxin B 1 (75 µg/kg per os) Lutein/Zeaxanthin cohort received LUT/ZEA ( 100 mg/kg per os) AFB 1 + LUT/ZEA (low dose) : received 75µg/kg of AFB 1 and 100 mg/kg of LUT/ZEA AFB 1 + LUT/ZEA (high dose) : received 75µg/kg of AFB 1 and 200 mg/kg of LUT/ZEA LUT/ZEA and AFB 1 were dissolved in corn oil and administered orally for twenty-eight (28) days. The doses were selected based on previous literature: for AFB 1 [ 29 – 31 ] were used; for LUT/ZEA, references [ 23 , 32 , 33 ] were used. The experimental protocol and treatment adhered to these previously reported dosages documented in the scientific literature. Sample collection On day 29, the rats were weighed using a U.S. Solid Digital Analytical Balance (USS-DBS16, Cleveland, OH, USA), and blood samples were obtained from each rat via the retro-orbital sinus. The experimental rats were euthanised, and the liver and kidney tissue were removed and weighed to estimate the liver and kidney coefficient, mathematically expressed as a percentage ratio of tissue weight to body weight. The blood was allowed to clot, then centrifuged at 3000 g for 10 minutes to obtain serum samples, which were frozen at -20°C until kidney and liver function assays were performed. The liver and kidney tissues obtained were rinsed in cold 1.15% aqueous potassium chloride, homogenised (Teflon homogeniser) in phosphate buffer (0.1 M, pH 7.4), and centrifuged for 10 min at 15000 rpm to obtain supernatant for biochemical assay. Biochemical assay Determination of serum enzyme biomarkers Serum samples were used to quantify creatinine, urea, high-density lipoprotein (HDL), total cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) using kits purchased from Randox Laboratories Limited (UK) according to the manufacturer's instructions. Protein determination in the sample The total protein content of the supernatants from liver and kidney homogenates was measured using the Lowry technique [ 34 ]. The tissue sample (7 µL) and distilled water (23 µL) were mixed in a microplate, followed by the addition of 150 µL of the alkaline CuSO₄ solution. The resulting mixture was allowed to settle at room temperature for ten minutes. After adding 15 µL of Folin-Ciocalteu solution, the mixture was incubated for 30 minutes at room temperature. Finally, the absorbance (750 nm) was acquired with a spectrophotometer against a reagent blank. Determination of the oxido-nitrosative stress The level of superoxide dismutase (SOD) activity was measured by the inhibition of adrenaline auto-oxidation described by Misra and Fridovich [ 35 ]. Briefly, 5 µL of the sample and blank, along with 250 µL of 0.05 M carbonate buffer (pH 10.2), were added to a plate reader, followed by the addition of 30 µL of freshly prepared adrenaline. Absorbance was measured for 3 minutes at 480 nm, every 30 seconds. Catalase (CAT) activity was measured using Clairborne's technique [ 36 ], with hydrogen peroxide (H 2 O 2 ) serving as the substrate. 25 µl of the sample and blank were mixed with 118 µl of a hydrogen peroxide solution (19 mM) in a plate reader, and the mixture was immediately read at 240 nm (1 min interval for 5 minutes). Reduced glutathione (GSH) was determined by applying the method of Jollow et al . [ 37 ]. After adding 80 µL of TCA, the mixture was combined with 80 µL of the sample and blank, vortexed, and then centrifuged at 4000 rpm for 5 minutes. Then, 50 µL of the TCA supernatant was combined with 150 µL of Ellman's reagent and incubated for 10 min at room temperature. The absorbance was then read at 412 nm using a plate reader. Glutathione- s -transferase (GST) activity was assessed using Habig's technique [ 38 ], which allowed the reaction to proceed for three minutes after the estimated medium was prepared. Readings were taken at 340 nm every 60 seconds and compared to the blank. Also, glutathione peroxidase (GPx) activity was biochemically assessed following the methods of Rotruck et al . [ 39 ]. A test tube was filled with 50 µL of phosphate buffer, 10 µL of NaN 3 , 20 µL of GSH, 10 µL of H 2 O 2 , and 50 µL of the sample, which was added last. After adding 50 µL of TCA and incubating for three minutes at 37˚C, the reaction mixture was centrifuged for five minutes at 3000 rpm. 100 µL of K 2 HPO 4 and 50 µL of DTNB were combined with 50 µL of the supernatant. Next, the absorbance was measured at 412 nm against a reagent blank that included 50 µL of DTNB, 100 µL of K 2 HPO 4 , and 50 µL of distilled water. The total sulfhydryl cohort (TSH) was determined using Ellman's method [ 40 ]. 150 µL of the sample, 100 µL of phosphate buffer, and 250 µL of distilled water were pipetted into an Eppendorf tube. The mixture was then left to stand. After pipetting 15 µL of Ellman's reagent into a microplate, 230 µL of the reaction mixture was added. The mixture was then left to stand for two minutes. At 412 nm, the absorbance was measured. Measurement of an inflammatory biomarker A pro-inflammatory biomarker, Xanthine oxidase (XO), was assessed by measuring XO activity as a biomarker of inflammation. Quantification of XO was performed using the method described by Bergmeyer et al . [ 41 ]. A 150-µL sample, 100 µL of phosphate buffer, and 250 µL of distilled water were pipetted into an Eppendorf tube. After that, the mixture was left to stand. After pipetting 15 µL of Ellman's reagent onto a microplate, 230 µL of the reaction mixture was added, and the mixture was left to stand for 2 minutes. At 412 nm, the absorbance was then measured. Meanwhile, the protocols of Green et al . [ 42 ] were utilised to estimate the amounts of nitric oxide (NO) for the NO assay. After the Griess reaction, the concentrations of nitrite in liver and kidney supernatants were determined by adding 100 µL of the samples to 100 µL of Griess reagent (0.1%) N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide in 5% phosphoric acid for 20 minutes at room temperature. The absorbance at 550 nm (OD 550) was measured and compared with a standard curve of sodium nitrite to estimate the nitrite concentration. Myeloperoxidase (MPO) activity was measured by modifying the technique outlined by Trush et al. [ 43 ]. 200 µL of O-dianisidine dihydrochloride, 50 µL of diluted H 2 O 2 , and 7 µL of tissue homogenate were added to a 96-well plate and read at 460 nm for four minutes, at 30-second intervals, expressed as units of MPO/mg tissue. Histopathology and Histomorphometry Evaluation The liver and Kidney tissues were excised immediately after sacrifice, rinsed in ice-cold saline and fixed in 10% formalin for 48 hours. Fixed tissues were dehydrated through graded ethanol (70–100%), cleared in xylene, and embedded in paraffin wax. Serial sections of 4–5 µm thickness were cut using a rotary microtome and mounted on glass slides. Sections were stained with hematoxylin and eosin (H&E) following standard protocols to visualize glomerular and tubular morphology The liver and kidney tissue (n = 3/group) were excised immediately after sacrifice, rinsed in ice-cold saline and fined in 10% formalin for 48 hours. The fixed tissues were dehydrated in graded ethanol (70–100%), cleared in xylene and embedded in paraffin wax. Microtome section (4–5 µm) were obtained and mounted on glass slides and stained with hematoxylin and eosin (H&E) [ 44 ]. histoarchitecture of the liver and kidney section were determined using light microscope at 400X and ImageJ software was used to obtain average hepatocyte count and average diameter of the glomerulus in five non-overlapping photomicrographs. Semi-quantitative scoring scale of tubular and interstitial lesion was performed on five non-overlapping photomicrographs/kidney tissue slide including tubular necrosis, tubular degeneration, tubular dilatation, basement membrane thickening and interstitial fibrosis [ 45 ]. Statistical Analysis The data obtained were normalised using the D'Argostino-Pearson Omnibus and Shapiro-Wilk tests and expressed as means ± standard deviation. The mean differences, analysed using one-way analysis of variance (ANOVA) followed by a post-hoc test (Tukey test), were analysed using GraphPad Prism (version 10) (CA, USA). The significance level was set at * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 . Results Network Pharmacology Potential targets of AFB 1 , hepatorenal toxicity, and LUT/ZEA were identified using online databases as mentioned earlier. Approximately 346 targets of “AFB 1 ,” 257 targets of “hepatorenal toxicity,” and 330 targets of LUT/ZEA were found. 13 intersection targets of AFB 1 , Hepatorenal Toxicity, and LUT/ZEA were collected for subsequent analysis. ALB, IL2, LGALS3, SRC, REN, EGFR, PRKACA, LCN2, F2, and TTR were the top 10 hub targets according to MCC values of AFB 1 -induced hepatorenal toxicity mitigated by LUT/ZEA. The PPI network for AFB 1 and hepatorenal toxicity had 22 nodes and 77 edges, while AFB 1 -induced hepatorenal toxicity mitigated by LUT/ZEA had 13 nodes and 30 edges, constructed using the intersection targets of AFB 1 , hepatorenal toxicity, and LUT/ZEA. The topological parameters of the PPI network were shown in Fig. 3 . In this study, there were 28 edges and 10 nodes in the PPI network of hub targets (Fig. 3 C). The enrichment analysis results were filtered using -log 10 (FDR), the top 20 terms of GO enrichment were selected and displayed in Fig. 3 . The findings demonstrated that the targets of AFB 1 -induced hepatorenal toxicity mitigated by LUT/ZEA involved biological processes such as hormone transport, response to stress, regulation of multicellular organismal-level homeostasis, regulation of body fluid level, hormone secretion and tissue remodelling. The targets were related to the cellular component (CC) in extracellular membrane-bound organelles, cytoplasmic vesicles, dendritic filopodia, etc. The molecular function (MF) terms of targets included protein kinase activity, molecular function regulator activity, epidermal growth factor receptor activity, ATPase binding, hormone binding, etc. The top 20 terms of KEGG pathways enrichment were selected and displayed in Fig. 3 G. The KEGG analysis showed that the targets of AFB 1 -induced hepatorenal toxicity were mainly enriched in bladder cancer, thyroid hormone synthesis, chemical carcinogenesis receptor activity, oxytocin signaling pathways, estrogen signaling pathway, and pathways in cancer. Molecular docking Molecular docking was applied to ALB, IL2, LGALS3, SRC, REN, EGFR, PRKACA, LCN2, F2, and TTR, and the docking interactions of these targets with AFB 1 , lutein, and zeaxanthin are presented in Figs. 5 – 9 . AFB 1 bound strongly to ALB through a carbon-hydrogen bond with Ala175, and pi-sigma interaction with Ala176. Lutein had a weak binding with ALB through alkyl interactions with Ala176, Lys519, Pro180, Lys436, Ala191, and Leu179. Zeaxanthin also bound weakly through polar interactions with Leu210, Leu264, and Val270 (Figs. 5A1, 5A2, 5A3). AFB 1 bound strongly to IL2 through conventional hydrogen bond with Arg120, carbon-hydrogen bonds with Glu116 and Thr111, and pi-sigma interaction with Met46. Lutein doesn’t bind strongly to ALB but forms an alkyl, pi-alkyl bond with His16, Ile122. Zeaxanthin had an unfavorable bump with Asn26, which shows a repulsion between zeaxanthin and the binding site of IL2 (Fig. 5B1-5B 3 ). AFB 1 bound strongly to IL2 through conventional hydrogen bond with Arg120, carbon-hydrogen bond with Glu116 and Thr111, and pi-sigma interaction with Met46. Lutein doesn’t bind strongly to ALB but forms an alkyl, pi-alkyl bond with His16, Ile122. Zeaxanthin had an unfavorable bump with Asn26, which shows a repulsion between zeaxanthin and the binding site of IL2 (Fig. 5B1- 5B 3 ). AFB 1 bound strongly to LGALS3 through conventional hydrogen interactions with HIS158 and Lys176, carbon-hydrogen interactions with Gly182 and Asn180, and pi-pi stacked interaction with Trp181. Lutein binds strongly to LGALS3 through conventional hydrogen interaction with Asn160, and pi-sigma interaction with Trp181. Zeaxanthin binds to LGALS3 through pi-sigma interactions with Trp181 (Fig. 6A1-6A 3 ). AFB 1 interacted with SRC through carbon carbon-hydrogen bond with Tyr479, a carbon-hydrogen bond with VAL461, and a pi-sulfur bond with Met481. Lutein interacted with SRC through pi-sigma bond with His492, unfavourable bonds with Glu505, Trp499, Met481, and Cys496. Zeaxanthin interacted with SRC through a conventional hydrogen bond with Glu505, and unfavourable bonds with Met481, Cys496, Thr453, and His492 (Fig. 6B1-6B 3 ). AFB 1 interacted with REN through a conventional hydrogen bond with Gly96 and Gly95, a carbon-hydrogen bond with Asn184 and Phe331, and a pi-pi T-shaped bond with Phe318. Lutein interacted with REN through a conventional hydrogen bond with Ser161 and Lys270. Zeaxanthin interacted with REN through a conventional hydrogen bond with Asp160 and Glu162, and an unfavourable bump bond with Tyr285 (Fig. 7A1-7A 3 ). AFB 1 interacted with EGFR through a conventional hydrogen bond with His209. Lutein interacted weakly with EGFR through alkyl bonds. Zeaxanthin interacted weakly with EGFR through an alkyl bond (Fig. 7B1-7B 3) . AFB 1 interacted with PRKACA through conventional hydrogen bond with Arg133, carbon-hydrogen bond with Pro236, pi-cation and pi-anion with Glu203 and Phe129. Lutein interacted with PRKACA through pi-sigma bond with Phe129 and alkyl bond with Ala240, Phe239, Phe240, Ala240 and Lys168. Zeaxanthin interacted with PRKACA through pi-sigma bond with Phe129, alkyl and pi-alkyl bond with Ala240, Phe239, Phe129, Lys168 (Fig. 8A1-8A 3 ). AFB 1 interacted with LCN2 through conventional hydrogen bonds with Tyr126, Ser88, Arg101, and pi-cation bonds with Lys154. Lutein interacted with LCN2 through a conventional hydrogen bond with Asp67 and Glu64, and pi-alkyl bond with Trp99 and Tyr120. Zeaxanthin interacted with LCN2 through carbon carbon-hydrogen bond with Pro68, and unfavourable bumps with Leu62, Gln69, and Val187 (Fig. 8B1-8B 3 ). AFB 1 interacted with F2 through a conventional hydrogen bond with Arg221 and Arg221 and pi-carbon bond with Arg173. His209. Lutein interacted with F2 through a conventional hydrogen bond with Gly186. Zeaxanthin interacted weakly with F2 through alkyl bonds (Fig. 9A1-9A 3 ). AFB 1 interacted with TTR through conventional hydrogen bond with Lys35, Trp41, and Lys70, carbon-hydrogen bond with Glu89 and His90, and pi-anion bond with Glu72. Lutein interacted with TTR through van der Waals bonds with His31, Asn74, Glu72, Lys70, and Ala29. Zeaxanthin interacted with TTR through a pi-alkyl bond with Trp41(Fig. 9B1-9B 3 ). Effect of Lutein/Zeaxanthin on Relative Organ Weight in AFB 1 Treated Rats. The effect of Lutein/Zeaxanthin ( LUT/ZEA) on the hepatorenal-somatic index of rats exposed to AFB 1 is depicted in Table 1 . At the end of the experimental period, rats exposed to AFB 1 had higher ( p < 0.05 ) relative liver and kidney weights compared to the control cohort. In contrast, the AFB 1 + LUT/ZEA 2 cohort (high dose; 200 mg/kg) showed increased body weight gain, whereas the low-dose cohort (100 mg/kg) did not, compared to the AFB 1 -only administered cohort. Table 1 Effect of the administration of Lutein/Zeaxanthin in Rats Treated Aflatoxin B 1 induced toxicity on the body weight, organ weight, and relative organ weight of rats Weights(g) Control AFB 1 Lut/Zea (100 mg/kg) AFB 1 + Lut/Zea (100 mg/kg) AFB 1 + Lut/Zea (200 mg/kg) Final Body weight 216.1 ± 32.92 204.8 ± 18.53 215.4 ± 20.06 211.1 ± 16.76 216.6 ± 8.69 Initial Body Weight 213.7 ± 25.61 201.1 ± 17.93 209.8 ± 18.23 206.8 ± 16.37 213.3 ± 10.59 Body Weight Gain 2.43 ± 14.98 3.63 ± 4.37 5.625 ± 5.181 4.38 ± 6.68 3.29 ± 6.39 Liver weight (g) 6.02 ± 0.78 5.53 ± 0.49 5.35 ± 1.22 5.18 ± 0.33 5.84 ± 0.70 Relative Liver weight 2.321 ± 1.19 2.70 ± 0.25 2.48 ± 0.62 2.46 ± 0.17 2.247 ± 1.15 Kidney weight (g) 1.26 ± 0.15 1.13 ± 0.19 1.133 ± 0.23 1.05 ± 0.12 1.06 ± 0.11 Relative Kidney weight 0.49 ± 0.25 0.55 ± 0.25 0.53 ± 0.25 0.49 ± 0.25 0.41 ± 0.25 Rats were administered with Aflatoxin B 1 (75µg/kg BW), Lutein/Zeaxanthin (100 mg/kg BW), Aflatoxin B 1 (75µg/kg per BW) plus Lutein/Zeaxanthin (100 and 200 mg/kg per BW), or vehicle for 28 consecutive days of feeding (n = 8 per group). Data are expressed as mean ± SD. Differences between the treatment groups were analyzed by student's t-test. AFB 1 : Aflatoxin B 1 ; LUT/ZEA: Lutein/Zeaxanthin. Table 2 Docking scores of the target genes with AFB 1, Lutein, and Zeaxanthin Target Genes PDB ID Docking Scores (Kcal/mol) AFB 1 LUT ZEA ALB 1AO6 -7.1 -6.8 -6.3 IL2 1M47 -5 -4.1 -2 LGALS3 3T1M -4.1 -9 -9.5 SRC 1FMK -4.1 -3 -2.5 REN 2I4Q -8.8 -9.5 -8.9 EGFR 4R3P -7.6 -6.6 -4.6 PRKACA 7Y1G -8.6 -5.4 -5.8 LCN2 3BX8 -7.9 -2.8 -3 F2 2AFQ -9.5 -6.3 -6.3 TTR 1DVQ -5.7 -5.2 -5.6 Table 3 Effect of the administration of Lutein/Zeaxanthin on semi-quantitative scoring of tubular and interstitial lesion in rats exposed to Aflatoxin B 1 Semi-quantitative scoring scale Control AFB 1 (75µg/kg) Lut/Zea (100 mg/kg) AFB 1 + Lut/Zea (100 mg/kg) AFB 1 + Lut/Zea (200 mg/kg) Tubular necrosis - +++ - + - Tubular Degeneration - +++ -/+ + -/+ Tubular Dilatation - +++ - + -/+ Basement membrane thickening -/+ +++ -/+ + -/+ Interstitial fibrosis - +++ - - - Rats were administered with Aflatoxin B 1 (75µg/kg BW), Lutein/Zeaxanthin (100 mg/kg BW), Aflatoxin B 1 (75µg/kg per BW) plus Lutein/Zeaxanthin (100 and 200 mg/kg per BW), or vehicle for 28 consecutive days of feeding (n = 3/ group). Semi-quantitative scoring scale is indicated by absence (-) or presence (+) of tubular or interstitial lesion. AFB 1 : Aflatoxin B 1 ; LUT/ZEA: Lutein/Zeaxanthin. Effects of LUT/ZEA on Liver and Kidney Function Biomarkers in AFB 1 Treated Rats The impact of co-exposure to AFB 1 and LUT/ZEA on the serum indicators of hepatic function is shown in Fig. 10 . Compared to the control cohort, AFB 1 -treatment significantly ( p < 0.05 ) increased serum AST [F (4, 15) = 35.55, P < 0.0001] and ALT [F (4, 15) = 12.58, P = 0.0001] activity, creatinine [F (4, 15) = 27.31, P < 0.0001] and urea [F (4, 15) = 11.92, P = 0.0001] level. Conversely, treatment with AFB 1 + LUT/ZEA (100 mg/kg and 200 mg/kg) caused a significant ( p < 0.05 ) reduction in AST and ALT activity, creatinine, and urea levels when compared to the AFB 1 -treated cohort. Effects of LUT/ZEA on Lipid Profile in AFB 1 Treated Rats Figure 11 outlines the effect of the co-exposure to AFB 1 and LUT/ZEA on lipid profile. Compared to the control cohort, the AFB 1 -treated cohort significantly increased total cholesterol (TC) [F (4, 15) = 15.25, P < 0.0001], triglyceride [F (4, 15) = 18.52, P < 0.0001] and decreased high-density lipoprotein level [F (4, 15) = 18.74, P < 0.0001]. At the same time, AFB 1 was co-treated with different doses of LUT/ZEA (100 mg/kg and 200 mg/kg), resulting in significantly lower TC and TG levels and increased HDL levels, compared to the AFB 1 -treated cohort. Effect of Lutein/Zeaxanthin on the Hepatorenal Antioxidant System in AFB 1 Treated Rats Figure 12 – 13 illustrates the impact of lutein and zeaxanthin on liver and kidney antioxidant biomarkers of rats exposed to 75 µg/mg AFB 1 . Experimental rats treated with AFB 1 alone showed significantly reduction ( p < 0.05 ) in the liver (upper panel) and the kidney (lower panel) SOD [F (4, 15) = 26.04, P < 0.0001; F (4, 15) = 67.54, P < 0.0001], CAT [F (4, 15) = 20.02, P < 0.0001], GPx [F (4, 15) = 35.23, P < 0.0001; F (4, 15) = 84.00, P < 0.0001], GST [F (4, 15) = 23.98P < 0.0001; F (4, 15) = 29.09, P < 0.0001; F (4, 15) = 12.22, P = 0.0001, GSH [F (4, 15) = 25.08, P < 0.0001; F (4, 15) = 12.22, P = 0.0001], and TSH [F (4, 15) = 9.732, P = 0.0004; F (4, 15) = 21.81, P < 0.0001] levels compared to the control cohort. On the other hand, in the administration of LUT/ZEA at doses of 100 mg/kg and 200 mg/kg, there was a significant increase in the antioxidant enzymatic activities. Specifically, there was an increase ( p < 0.05 ) in liver and kidney SOD and CAT activities at both doses, 100 mg/kg and 200 mg/kg. Effect of Lutein/Zeaxanthin on the hepatorenal oxido-inflammatory responses in AFB1-treated rats Figure 14 presents the effect of LUT/ZEA treatment on the mediators of oxidative stress and inflammatory response assessed in the liver (upper panel) and kidney (lower panel) of rats exposed to AFB 1 . Compared to the control cohort, the AFB 1 -treated cohort significantly increased hepatorenal XO [F (4, 15) = 24.52, P < 0.0001; F (4, 15) = 28.94, P < 0.0001], NO [F (4, 15) = 18.04, P < 0.0001; F (4, 15) = 33.95, P < 0.0001], and MPO [F (4, 15) = 28.36, P < 0.0001; F (4, 15) = 25.82, P < 0.0001] activity compared to the control. On the other hand, AFB 1 + LUT/ZEA (100 mg/kg and 200 mg/kg) significantly ( p < 0.05 ) decreased liver and kidney XO, NO, and MPO activities at 100 mg/kg, and a significant ( p < 0.05 ) decrease in these activities at 200 mg/kg. Effect of Lutein/Zeaxanthin on the hepatorenal tumor and apoptotic biomarkers in AFB 1 -treated rats Apoptosis biomarkers were further assessed to investigate the impact of LUT/ZEA in rats exposed to AFB 1 (Fig. 15 ) . Rats exposed to AFB 1 had higher TP53 [F (4, 10) = 56.12, P < 0.0001, F (4, 10) = 15.36, P = 0.0003], BAX [F (4, 10) = 20.63, P < 0.0001; F (4, 10) = 16.25, P = 0.0002] and reduced Bcl2 [F (4, 10) = 13.43; F (4, 10) = 7.291, P = 0.0051] concentrations and an increase in BAX/Bcl2 ratio [F (4, 10) = 82.29, P < 0.0001; F (4, 10) = 20.40, P < 0.0001] than control rats. However, rats co-treated with AFB 1 + LUT/ZEA-treated rats demonstrated a substantial decrease in TP53, BAX, and reduced Bcl2 level, which increased compared to AFB 1 -only treated rats. Effect of Lutein/Zeaxanthin on the hepatorenal histological architecture in AFB 1 -treated rats H&E microscopic examination of the liver and kidney are illustrated in Figs. 16 and 17 . In the liver section, control and LUT/ZEA only treated rats reveal normal histopathology indicated by significant increase in viable hepatocyte count. AFB 1 -only treated rats demonstrated decrease in hepatocyte count [F (4, 10) = 31.13, P < 0.0001], inflammatory cell infiltration, necrosis. AFB 1 + LUT/ZEA-treated rats confirmed alleviation specifically at dose of 200 mg/kg compered to AFB 1 -only treated rats. in the kidney, control and LUT/ZEA only treated rats revealed normal glomeruli, the absences of tubular necrosis, tubular degeneration, tubular dilatation, basement membrane thickening and interstitial fibrosis. Experimental rat treated with AFB 1 -only showed reduction in glomeruli [F (4, 10) = 21.35, P < 0.0001], obliterated capillary structure and inflammatory cell infiltration and notable tubular necrosis, tubular degeneration, tubular dilatation, basement membrane thickening and interstitial fibrosis. AFB 1 + LUT/ZEA-treated rats at 100 and 200 mg/kg reversed glomeruli and tubular lesion caused by AFB 1 -only treatment. Discussion This study provides an integrated assessment of the protective potential of lutein and zeaxanthin (LUT/ZEA) against Aflatoxin B 1 (AFB 1 )-induced hepatorenal toxicity, combining network pharmacology, molecular docking, and in vivo validation. The findings contribute valuable insights into the molecular mechanisms underlying LUT/ZEA’s mitigating effects, highlighting their promise as multi-targeted agents for alleviating mycotoxin-induced organ injury. AFB 1 is recognized as a potent hepatotoxin and nephrotoxin, with a well-documented ability to induce oxidative stress, inflammation, apoptosis, and mitochondrial dysfunction [ 31 ] and likely deleterious mutations in sites where AFB 1 causes DNA damage [ 31 , 46 ], These processes culminate in cellular damage and increase the risk of carcinogenesis, as supported by previous literature and reinforced by the current results.. Despite increasing knowledge of AFB 1 ’s multi-organ toxicity, therapeutic interventions remain limited, underscoring the need for novel protective strategies. Recent epidemiological and experimental evidence suggests that higher dietary intakes of carotenoids are associated with a reduced risk of liver disease, particularly non-alcoholic fatty liver disease (NAFLD)[ 47 , 48 ]. Lutein and zeaxanthin, notable for their antioxidant, anti-inflammatory, and anti-apoptotic properties, have demonstrated organ-protective effects in various tissues, including the eye, brain, heart, skin, and liver. Network pharmacology analyses offered further mechanistic understanding by identifying a set of central hub genes (ALB, EGFR, IL2, REN, LCN2, SRC, LGALS3, PRKACA, F2, TTR) involved in AFB1-related hepatorenal toxicity. The enrichment of molecular functions related to epidermal growth factor receptor activity and kinase signaling, particularly involving EGFR and SRC, connects these targets to cellular proliferation, survival, and stress responses. Moreover, involvement of interleukin-2 (IL-2) receptor binding points to the role of LUT/ZEA in modulating immune and inflammatory cascades, although the lower binding affinity for IL-2 compared to AFB 1 suggests limited efficacy in suppressing certain inflammatory pathways. GO biological process enrichment underscored the negative regulation of mitochondrial depolarization as a core protective mechanism. Since mitochondrial dysfunction is central to AFB1-induced cytotoxicity, the ability of LUT/ZEA to stabilize cellular bioenergetics reflects a crucial aspect of their protective profile. KEGG pathway analyses further highlighted the relevance of EGFR tyrosine kinase inhibitor resistance, ErbB signaling, and renin secretion pathways—linking these findings directly to mechanisms of chronic renal injury and carcinogenesis. Molecular docking simulations revealed that LUT/ZEA exhibited strong binding affinities for several key hub proteins, notably REN and LGALS3, surpassing AFB 1 in binding strength for these targets. This implies that LUT/ZEA may function as effective inhibitors of pro-inflammatory, proliferative, and fibrotic pathways. The ability of zeaxanthin and lutein to bind more effectively to LGALS3, a principal mediator of fibrosis, and REN, a regulator of the renin-angiotensin-aldosterone system, provides a molecular basis for their observed systemic protective effects. LUT/ZEA’s interactions with LCN2 and F2 further support their role in attenuating acute kidney injury and hepatic complications. The present study demonstrated that LUT/ZEA administration in a rat model exposed to AFB 1 resulted in notable reductions in biochemical markers indicative of liver damage (AST and ALT, which play roles in amino acid metabolism and are released when the liver is damaged) - and kidney (urea and creatine) injury [ 11 , 20 ]. Improvements in serum levels of ALT, AST, creatinine, and urea indicate that LUT/ZEA effectively mitigate AFB 1 -induced tissue injury. These results support earlier findings [ 21 , 22 ] and align with the rise observed in the hepatorenal somatic index, reflecting injury following AFB 1 toxicity. However, rats treated with LUT/ZEA exhibited reversed effects, demonstrated by lower serum levels of AST, ALT, creatinine, and urea, implying a protective role against liver and kidney damage. Notably, the marked reduction in enzyme activity and metabolic waste in the bloodstream suggests this protective effect against AFB 1 compromised liver and kidneys structural integrity. This biochemical improvement is corroborated by histopathological findings, which demonstrates that LUT/ZEA treatment attenuated hepatocyte necrosis, inflammatory cell infiltration as well as glomeruli and tubular degeneration and interstitial fibrosis. The liver is essential for energy and lipid metabolism, and signs of liver damage are often linked to lipid buildup [ 49 ]. The present results indicate that AFB 1 disrupts lipid metabolism, as evidenced by higher triglyceride (TG) and total cholesterol (TC) levels, along with reduced high-density lipoprotein (HDL)—known as good cholesterol—signalling dyslipidemia, a common marker of hepatic parenchymal cell harm [ 25 , 26 ]. Furthermore, LUT/ZEA supplementation enhanced lipid profiles, reducing triglycerides and total cholesterol while increasing high-density lipoprotein levels, thereby counteracting dyslipidemia, a common consequence of hepatic damage. This observation supports prior research suggesting LUT/ZEA as an effective intervention for dyslipidemia, especially in older adults [ 27 , 50 ]. AFB 1 -related hepatorenal injury results in an overproduction of free radicals and oxidative stress, which plays a key role in AFB 1 potent carcinogenicity [ 51 ]. These free radicals can harm cell membranes by damaging biomolecules such as proteins, lipid components, and DNA, leading to lipid peroxidation, reduced endogenous antioxidant activity, and a loss of cellular function and structural integrity [ 52 ]. However, supplementation with LUT/ZEA has been associated with a reduction in oxidative stress [ 53 ]. This protective effect is connected to increased antioxidant activity, including superoxide dismutase (SOD), catalase (CAT), the glutathione system (GPx, GSH, GST), and total sulfhydryl (TSH)—which neutralise harmful reactive oxygen species, like superoxide radicals and hydrogen peroxide (H₂O₂), converting them into harmless substances such as water and oxygen via the Fenton reaction. Evidence shows that AFB 1 causes oxidative stress in liver and kidney cells by accumulating free radicals, contributing to lipid peroxidation and tissue damage [ 54 , 55 ]. Our findings indicate that LUT/ZEA boosts hepatic and renal antioxidant activities, shown by higher levels of SOD, CAT, GPx, GSH, and GST, aligning with previous research [ 56 , 57 ] and supporting their role in protecting cell membranes from oxidative injury in the liver and kidneys [ 58 – 61 ]. Additionally, rats cotreated with LUT/ZEA displayed xanthine oxidase (XO) inhibitory activity, likely due to their molecular structure’s rich in conjugated double bonds, enabling effective scavenging of free radicals and inhibition of oxidative enzymes like XO [ 20 ]. Our results further demonstrated that administration of LUT/ZEA at doses of 100 and 200 mg/kg led to a reduction in inflammatory mediators, as indicated by decreased nitric oxide (NO) levels and myeloperoxidase (MPO) activity, both of which were significantly increased in rats exposed to AFB 1 . This suggests that LUT/ZEA may confer protection against NO production, thereby limiting the formation of peroxynitrite, a highly reactive and deleterious oxidant [ 62 ]. Additionally, the observed decrease in neutrophil count may have mitigated cell destruction, a pivotal component in the inflammatory and immune response to tissue injury [ 63 , 64 ]. Considering the ameliorative effects of LUT/ZEA on AFB 1 -induced inflammation and oxidative damage, we extended our investigation to examine their influence on apoptotic biomarker modulation following AFB 1 exposure. Apoptosis, a key contributor to liver and kidney injury, [ 65 – 69 ]. AFB 1 has been shown to induce hepatic [ 70 – 72 ] and renal [ 73 – 75 ] apoptosis by downregulating anti-apoptotic protein Bcl2 and upregulating pro-apoptotic proteins BAX and TP53. These biomarkers are indicative of the activation of multiple apoptotic pathways, including cytochrome c release and the subsequent activation of caspases 3 and 9 through mitochondrial signalling [ 14 , 76 ]. Apoptosis was also ameliorated by LUT/ZEA supplementation. The suppression of pro-apoptotic markers and the preservation of anti-apoptotic proteins suggest that LUT/ZEA help maintain the integrity and function of hepatic and renal tissues in the face of AFB 1 challenge. Taken together, these findings establish LUT/ZEA as promising candidates for the prevention or amelioration of AFB 1 -induced hepatorenal toxicity. Their multifaceted mechanisms—encompassing modulation of oxidative stress, inflammation, apoptosis, and key cellular pathways—make them attractive for further investigation as dietary or therapeutic interventions, particularly in populations at risk for chronic mycotoxin exposure. Nevertheless, the study’s reliance on histopathological and biochemical endpoints indicates a need for broader molecular investigations. Future research should explore detailed signaling networks and validate findings in human subjects to optimize dosing and assess long-term safety. In conclusion, this study advances our understanding of the protective roles of lutein and zeaxanthin in counteracting the toxic effects of AFB 1 on liver and kidney tissues. The integration of network pharmacology, molecular docking, and in vivo experimentation provides a robust framework for elucidating their therapeutic potential and supports the ongoing pursuit of effective interventions against mycotoxin-induced organ damage. Abbreviations FDR False Discovery Rate LUT/ZEA Lutein/Zeaxanthin AFB 1 Aflatoxin B 1 RAAS Renin-Angiotensin-Aldosterone System ALB Albumin IL2 Interleukin 2 LDALS3 Galectin 3 SRC Proto-oncogene, non-receptor tyrosine kinase REN Renin EGFR Epidermal Growth Factor Receptor PRKACA cAMP-dependent protein kinase catalytic subunit alpha LCN2 Lipocalin 2 F2 Coagulation factor 2 TTR Transthyretin gene Declarations Acknowledgement We thank the following students for their technical support: Japheth Auta, Praise Dyap, Marvellous Salami, Mark Nnamdi, Dooshima Bagu, Precious Taiye, Abisola Ibukunle, and Oluwadunsin Adekunle. Ethical statement The protocols for the care and use of experimental animals in this study were approved by the University of Ibadan, Animal Care and Use in Research Ethical Committee, with approval number: Authors Contribution All authors participated in the design, interpretation, and analysis of the study's data. SO conceptualised the study; SO, JAO, JC, JCN and OO conducted the research and analysed the preliminary data. SO supervised the investigation, and SO, JAO, JOB, VOE, HA, NHS, OO and OO Proof checked the data for errors. EMP, SAA, IOA, VOE, HA, NHS and OO conducted the *in silico* study. SO, JC, JAO, JOB, IOA, VOE, HA, EMP, NHS, OO and OO wrote and revised the manuscript. Disclosure statement No potential conflict of interest was reported by the author(s). Availability of Data and Materials The datasets used and analysed during the current study are available from the corresponding author upon reasonable request. Funding The authors privately funded this research through their contributions and received no external grants from funding agencies in the commercial, not-for-profit, or public sectors. 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Ecotoxicol Environ Saf 269:115742 Xu Q et al (2020) Critical role of caveolin-1 in aflatoxin B1-induced hepatotoxicity via the regulation of oxidation and autophagy. Cell Death Dis 11(1):6 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Mar, 2026 Reviewers invited by journal 18 Dec, 2025 Editor assigned by journal 15 Dec, 2025 Submission checks completed at journal 15 Dec, 2025 First submitted to journal 11 Dec, 2025 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. 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1","display":"","copyAsset":false,"role":"figure","size":519372,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental protocol of Aflatoxin B\u003csub\u003e1\u003c/sub\u003e and LUT/ZEA co-exposure to adult male Wistar rats for 28 consecutive days. Created in BioRender. Abdullai Sanusi. (2025). \u003ca href=\"https://BioRender.com\"\u003ehttps://BioRender.com\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/ad2470aaec1417f81d4c04d9.png"},{"id":99313631,"identity":"ef041194-ebae-44b9-b178-93053086fbf7","added_by":"auto","created_at":"2025-12-31 16:20:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":813715,"visible":true,"origin":"","legend":"\u003cp\u003eProtein-protein interaction (PPI) network of genes, and hub genes identification in AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatotoxicity and LUT/ZEA-induced mitigation. \u003cstrong\u003eA\u003c/strong\u003e: Venn diagram showing the overlap between genes related to aflatoxin B\u003csub\u003e1\u003c/sub\u003e(AFB\u003csub\u003e1\u003c/sub\u003e) and hepatorenal toxicity (HR Toxicity). \u003cstrong\u003eB\u003c/strong\u003e: Interaction between the target genes of AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatorenal toxicity. \u003cstrong\u003eC\u003c/strong\u003e: Venn diagram showing the overlap between genes related to aflatoxin B\u003csub\u003e1\u003c/sub\u003e (AFB\u003csub\u003e1\u003c/sub\u003e), hepatorenal toxicity (HR Toxicity) and lutein/zeaxanthin (LUT/ZEA). \u003cstrong\u003eD\u003c/strong\u003e: Interaction between the target genes of LUT/ZEA-induced mitigation. The interaction networks illustrate how the genes are linked to one another.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/5736a102b50e8cc062da08f1.png"},{"id":99312711,"identity":"6faeb176-6c14-437c-b972-84db382c8061","added_by":"auto","created_at":"2025-12-31 16:19:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1116736,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of potential targets of AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatorenal toxicity and LUT/ZEA-mitigated AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatorenal toxicity, construction of PPI network and enrichment analysis. \u003cstrong\u003eA\u003c/strong\u003e: PPI network of the target genes of AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatotoxicity. \u003cstrong\u003eB\u003c/strong\u003e: MCC was used to construct the top 13 hub genes involved in LUT/ZEA-induced mitigation of AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatotoxicity. \u003cstrong\u003eC\u003c/strong\u003e: Top target clustering of LUT/ZEA-mitigated AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatotoxicity. The node size from large to small indicates that the MCC ranking values of the targets are from large to small in clockwise motion, and the colour difference is specific to individual targets. \u003cstrong\u003eD-E\u003c/strong\u003e: Gene ontology (GO) and pathway enrichment functional analysis of LUT/ZEA-mitigated AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatotoxicity targets with a focus on their biological processes (\u003cstrong\u003eD\u003c/strong\u003e), cellular components (\u003cstrong\u003eE\u003c/strong\u003e), molecular functions (\u003cstrong\u003eF\u003c/strong\u003e), and KEGG pathways (\u003cstrong\u003eG\u003c/strong\u003e). The size of the dots indicates the count of enriched genes, and the colour of the dot indicates the significance of the fold enrichment and false discovery rate (FDR), where red represents the most significant in all panels.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/1a105094adb778bb967212b8.png"},{"id":99312692,"identity":"d617a928-c9f7-44d7-b6eb-b87a7690fdcb","added_by":"auto","created_at":"2025-12-31 16:19:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":500652,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG pathway showing the genes involved in chemical carcinogenesis-receptor activation pathways. Red boxes highlight the genes impacted by LUT/ZEA these pathways.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/574364ed24e2192ba986c760.png"},{"id":99010005,"identity":"16c8aa85-f117-4b48-8a93-6d10e4dc9ed7","added_by":"auto","created_at":"2025-12-25 20:24:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1258218,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the binding modes of AFB\u003csub\u003e1\u003c/sub\u003e, Lutein, and Zeaxanthin with ALB and IL2. The 3D and 2D binding nodes of AFB\u003csub\u003e1\u003c/sub\u003e and ALB, LUT and ALB, and ZEA and ALB respectively (\u003cstrong\u003eA1-A3\u003c/strong\u003e). The 3D and 2D binding nodes of AFB\u003csub\u003e1\u003c/sub\u003e and IL2, LUT and IL2, and ZEA and IL2 respectively(\u003cstrong\u003eB1-B3\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/af441676702c732f9cde22f9.png"},{"id":99312888,"identity":"baf7e981-3c27-41ee-9692-ead589e612d7","added_by":"auto","created_at":"2025-12-31 16:19:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1261352,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the binding modes of AFB\u003csub\u003e1\u003c/sub\u003e, Lutein and Zeaxanthin with LGALS3 and SRC. The 3D and 2D binding nodes of AFB\u003csub\u003e1\u003c/sub\u003e and LGALS3, LUT and LGALS3, and ZEA and LGALS3 respectively (\u003cstrong\u003eA1-A3\u003c/strong\u003e). The 3D and 2D binding nodes of AFB\u003csub\u003e1\u003c/sub\u003e and SRC, LUT and SRC, and ZEA and SRC respectively(\u003cstrong\u003eB1-B3\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/d73032bcfb01f7f433489c81.png"},{"id":99313693,"identity":"03177367-9eee-4ef3-946a-37a68ad9a715","added_by":"auto","created_at":"2025-12-31 16:20:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1358041,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the binding modes of AFB\u003csub\u003e1\u003c/sub\u003e, Lutein and Zeaxanthin with REN and EGFR. The 3D and 2D binding nodes of AFB\u003csub\u003e1\u003c/sub\u003e and REN, LUT and REN, and ZEA and REN respectively (\u003cstrong\u003eA1-A3\u003c/strong\u003e). The 3D and 2D binding nodes of AFB\u003csub\u003e1\u003c/sub\u003e and EGFR, LUT and EGFR, and ZEA and EGFR, respectively(\u003cstrong\u003eB1-B3\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/e862195269f92f05b04a48a7.png"},{"id":99313251,"identity":"1036894a-ab89-4511-83c1-26da0293365d","added_by":"auto","created_at":"2025-12-31 16:19:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1092520,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the binding modes of AFB\u003csub\u003e1\u003c/sub\u003e, Lutein, and Zeaxanthin with PRKACA and LCN2. The 3D and 2D binding nodes of AFB\u003csub\u003e1\u003c/sub\u003e and PRKACA, LUT and PRKACA, and ZEA and PRKACA, respectively (\u003cstrong\u003eA1-A3\u003c/strong\u003e). The 3D and 2D binding nodes of AFB\u003csub\u003e1\u003c/sub\u003e and LCN2, LUT and LCN2, and ZEA and LCN2, respectively (\u003cstrong\u003eB1-B3\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/72456f5e558cfcd5fd2e03f6.png"},{"id":99010030,"identity":"1d9c8e61-2906-47d3-ab1e-6d7dbf21719a","added_by":"auto","created_at":"2025-12-25 20:24:35","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1183993,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the binding modes of AFB\u003csub\u003e1\u003c/sub\u003e, Lutein and Zeaxanthin with F2 and TTR. The 3D and 2D binding nodes of AFB\u003csub\u003e1\u003c/sub\u003e and F2, LUT and F2, and ZEA and F2, respectively (\u003cstrong\u003eA1-A3\u003c/strong\u003e). The 3D and 2D binding nodes of AFB\u003csub\u003e1\u003c/sub\u003e and TTR, LUT and TTR, and ZEA and TTR, respectively (\u003cstrong\u003eB1-B3\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/3cd21e37f8eba3e98b535dff.png"},{"id":99313255,"identity":"77c0c9c0-78da-4095-baad-1d9c1638893e","added_by":"auto","created_at":"2025-12-31 16:19:55","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":342994,"visible":true,"origin":"","legend":"\u003cp\u003eThe outcome of LUT/ZEA treatment on liver and kidney function tests of AFB\u003csub\u003e1\u003c/sub\u003e-treated rats for twenty-eight consecutive days. Control, (2 mL/kg); AFB\u003csub\u003e1\u003c/sub\u003e, 75 µg/kg; LUT/ZEA, 100 mg/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e1\u003c/sub\u003e, (75 µg+100mg)/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e2\u003c/sub\u003e, (75 µg+200 mg)/kg. Values are expressed as mean ± SD for five rats per cohort. Connecting lines indicate cohorts compared to one another, and the significance level was set at\u0026nbsp;\u003cem\u003e(p\u0026lt;0.05)\u003c/em\u003e; * to ****: indicates the significance level; ns: not significant. AFB\u003csub\u003e1\u003c/sub\u003e; Aflatoxin B\u003csub\u003e1\u003c/sub\u003e, LUT/ZEA; Lutein/Zeaxanthin; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase, creatinine, urea\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/2880feaf64e07fba88f5472e.png"},{"id":99312511,"identity":"566a8c6d-a55e-4ec0-bbb0-6c0b039dfac4","added_by":"auto","created_at":"2025-12-31 16:19:02","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":312606,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of treatment of LUT/ZEA on the lipid profile of AFB\u003csub\u003e1\u003c/sub\u003e-treated rats for twenty-eight consecutive days. Control, (2 mL/kg); AFB\u003csub\u003e1\u003c/sub\u003e, 75 µg/kg; LUT/ZEA, 100 mg/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e1\u003c/sub\u003e, (75 µg+100mg)/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e2\u003c/sub\u003e, (75 µg+200 mg)/kg. Values are expressed as mean ± SD for five rats per cohort. Connecting lines indicate cohort compared to one another, and the significance level was set at\u0026nbsp;\u003cem\u003e(p\u0026lt;0.05)\u003c/em\u003e; * to ****: indicates the significance level; ns: not significant. AFB\u003csub\u003e1\u003c/sub\u003e; Aflatoxin B\u003csub\u003e1\u003c/sub\u003e, LUT/ZEA; Lutein/Zeaxanthin;\u0026nbsp; TC: Total cholesterol; TG: Triglycerides; HDL: High-density lipoprotein.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/f178d7f10acc7f7694b84748.png"},{"id":99313930,"identity":"dbbb0f7a-4e4c-4986-915e-4fd79a956bb8","added_by":"auto","created_at":"2025-12-31 16:20:37","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":544273,"visible":true,"origin":"","legend":"\u003cp\u003eThe outcome of treatment of LUT/ZEA on activities of liver and kidney SOD, CAT and GPx of AFB\u003csub\u003e1\u003c/sub\u003e-treated rats for twenty-eight consecutive days. Control, (2 mL/kg); AFB\u003csub\u003e1\u003c/sub\u003e, 75 µg/kg; LUT/ZEA, 100 mg/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e1\u003c/sub\u003e, (75 µg+100mg)/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e2\u003c/sub\u003e, (75 µg+200 mg)/kg. Values are expressed as mean ± SD for five rats per cohort. Connecting lines indicate cohorts compared to one another, and the significance level was set at (\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e); * to ****: indicates the significance level; ns: not significant. AFB\u003csub\u003e1\u003c/sub\u003e, Aflatoxin B\u003csub\u003e1;\u003c/sub\u003e LUT/ZEA, Lutein/Zeaxanthin; SOD: Superoxide dismutase; CAT: Catalase, GPx: Glutathione peroxidase.\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/cc5d56dfaf7b90bf283c7b80.png"},{"id":99010025,"identity":"1824f2d5-8988-4c74-9e9f-16b85700a2ec","added_by":"auto","created_at":"2025-12-25 20:24:34","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":588402,"visible":true,"origin":"","legend":"\u003cp\u003eThe outcome of treatment of LUT/ZEA on the activities of liver and kidney GSH, GST and TSH of AFB\u003csub\u003e1\u003c/sub\u003e-treated rats for twenty-eight consecutive days. Control, (2 mL/kg); AFB\u003csub\u003e1\u003c/sub\u003e, 75 µg/kg; LUT/ZEA, 100 mg/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e1\u003c/sub\u003e, (75 µg+100mg)/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e2\u003c/sub\u003e, (75 µg+200 mg)/kg. Values are expressed as mean ± SD for five rats per cohort. Connecting lines indicate cohorts compared to one another, and the significance level was set at (\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e); * to ****: indicates the significance level; ns: not significant. AFB\u003csub\u003e1\u003c/sub\u003e, Aflatoxin B\u003csub\u003e1;\u003c/sub\u003e LUT/ZEA, Lutein/Zeaxanthin; GSH: Reduced glutathione; TSH: Total sulfhydryl cohort, and GST: Glutathione-\u003cem\u003es\u003c/em\u003e-transferase.\u003c/p\u003e","description":"","filename":"Figure13.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/3b21d6c94f9b1c3bf4106f2c.png"},{"id":99010050,"identity":"ddb67466-35f1-4334-9ac7-360df2044e34","added_by":"auto","created_at":"2025-12-25 20:24:35","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":563901,"visible":true,"origin":"","legend":"\u003cp\u003eThe outcome of LUT/ZEA treatment on liver and kidney oxido-inflammatory responses. Control, (2 mL/kg); AFB\u003csub\u003e1\u003c/sub\u003e, 75 µg/kg; LUT/ZEA, 100 mg/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e1\u003c/sub\u003e, (75 µg+100mg)/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e2\u003c/sub\u003e, (75 µg+200 mg)/kg. Values are expressed as mean ± SD for eight rats per cohort. Connecting lines indicate cohort compared to one another, and the significance level was set at (\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e); * to ****: indicates the significance level; ns: not significant. AFB\u003csub\u003e1\u003c/sub\u003e, Aflatoxin B\u003csub\u003e1;\u003c/sub\u003e LUT/ZEA, Lutein/Zeaxanthin; XO: Xanthine oxidase, NO: Nitric oxide; MPO: Myeloperoxidase.\u003c/p\u003e","description":"","filename":"Figure14.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/1924abe984f8ed130068393d.png"},{"id":99010012,"identity":"519af2f6-09b6-456b-8103-1f4fdf87a37d","added_by":"auto","created_at":"2025-12-25 20:24:34","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":547528,"visible":true,"origin":"","legend":"\u003cp\u003eThe outcome of treatment on the apoptotic biomarkers of the liver and kidney. Control, (2 mL/kg of corn oil); AFB\u003csub\u003e1\u003c/sub\u003e, 75 µg/kg; LUT/ZEA, 100 mg/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e1\u003c/sub\u003e, (75 µg+100mg)/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e2\u003c/sub\u003e, (75 µg+200 mg)/kg. Values are expressed as mean ± SD for eight rats per cohort. Connecting lines indicate cohort compared to one another, and the significance level was set at (\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e); * to ****: indicates the significance level; ns: not significant. AFB\u003csub\u003e1\u003c/sub\u003e, Aflatoxin B\u003csub\u003e1;\u003c/sub\u003e LUT/ZEA, Lutein/Zeaxanthin; TP53, Tumor Suppressor; BAX, Bcl-2 associated protein X; Bcl-2, B cell lymphoma 2.\u003c/p\u003e","description":"","filename":"Figure15.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/c966cef51099db7cbbc21efd.png"},{"id":99313497,"identity":"3131cc0c-d6a5-4ab0-85ed-c1c447d31c86","added_by":"auto","created_at":"2025-12-31 16:20:14","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":3791082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological findings and histomorphometrical measurements of the liver \u003c/strong\u003eControl, (2 mL/kg of corn oil); AFB\u003csub\u003e1\u003c/sub\u003e, 75 µg/kg; LUT/ZEA, 100 mg/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e1\u003c/sub\u003e, (75 µg+100mg)/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e2\u003c/sub\u003e, (75 µg+200 mg)/kg. The black arrow indicates the central portal triad (CPT), while the green arrow indicates the neutrophils. Values are expressed as mean ± SD for eight rats per cohort. Connecting lines indicate cohort compared to one another, and the significance level was set at (\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e); * to ****: indicates the significance level; ns: not significant. AFB\u003csub\u003e1\u003c/sub\u003e, Aflatoxin B\u003csub\u003e1;\u003c/sub\u003e LUT/ZEA, Lutein/Zeaxanthin.\u003c/p\u003e","description":"","filename":"Figure16.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/aea40b890ec29de50f7ae378.png"},{"id":99313650,"identity":"3b98af49-db82-400e-b94d-0260f25fcfb9","added_by":"auto","created_at":"2025-12-31 16:20:23","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":4007322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological findings and histomorphometrical measurements of the kidney. \u003c/strong\u003eControl, (2 mL/kg of corn oil); AFB\u003csub\u003e1\u003c/sub\u003e, 75 µg/kg; LUT/ZEA, 100 mg/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e1\u003c/sub\u003e, (75 µg+100mg)/kg; AFB\u003csub\u003e1\u003c/sub\u003e+LUT/ZEA\u003csub\u003e2\u003c/sub\u003e, (75 µg+200 mg)/kg. The black arrow indicates inflammatory cell infiltration, G; glomerulus, DCT, distal convoluted tubule. Values are expressed as mean ± SD for eight rats per cohort. Connecting lines indicate cohort compared to one another, and the significance level was set at (\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e); * to ****: indicates the significance level; ns: not significant. AFB\u003csub\u003e1\u003c/sub\u003e, Aflatoxin B\u003csub\u003e1;\u003c/sub\u003e LUT/ZEA, Lutein/Zeaxanthin.\u003c/p\u003e","description":"","filename":"Figure17.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/9e939384d51f71c38d88d46c.png"},{"id":99313051,"identity":"3b59e7e1-d7f4-4d37-8ef7-31127cbd3b1f","added_by":"auto","created_at":"2025-12-31 16:19:44","extension":"png","order_by":18,"title":"Figure 18","display":"","copyAsset":false,"role":"figure","size":852322,"visible":true,"origin":"","legend":"\u003cp\u003eAn experimental rat model's proposed mechanism of LUT/ZEA to mitigate AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatotoxicity in male Wistar rats. Proposed Mechanistic Insights into the Protective Effects of Lutein and Zeaxanthin Against Aflatoxin B\u003csub\u003e1\u003c/sub\u003e-Induced Liver and Kidney Toxicity in Rats Aflatoxin B\u003csub\u003e1\u003c/sub\u003e (AFB\u003csub\u003e1\u003c/sub\u003e), a mycotoxin from Aspergillus species, contaminates food supplies, especially in humid climates—and is linked to liver and kidney toxicity through oxidative stress, inflammation, DNA damage, and disrupted cell function. Researchers are exploring natural compounds for protection, with lutein and zeaxanthin—antioxidant carotenoids found in green vegetables, corn, and eggs—showing promise. These xanthophylls help counteract AFB\u003csub\u003e1\u003c/sub\u003e's toxic effects by scavenging reactive oxygen species (ROS) and boosting cellular antioxidant enzymes, restoring redox balance and protecting tissue structure. They also suppress pro-inflammatory cytokines (like TNF-α, IL-1β, IL-6) by inhibiting NF-κB signaling, thereby reducing inflammation and supporting organ recovery. Importantly, lutein and zeaxanthin defend against DNA adduct formation by neutralizing ROS and may enhance DNA repair mechanisms. They further regulate cell survival by inhibiting pro-apoptotic proteins (e.g., Bax) and increasing anti-apoptotic proteins (e.g., Bcl-2). Collectively, these actions provide multi-level protection of rat liver and kidneys against AFB\u003csub\u003e1\u003c/sub\u003e, suggesting lutein and zeaxanthin could be effective dietary interventions for populations at risk of aflatoxin exposure.\u0026nbsp; Created in BioRender. Abdullai Sanusi. (2025). \u003ca href=\"https://BioRender.com\"\u003ehttps://BioRender.com\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"Figure18.png","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/7ccb850163eacc9519caf792.png"},{"id":99787924,"identity":"9e36a777-ffb6-457f-8788-3bc040466585","added_by":"auto","created_at":"2026-01-08 12:41:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21193328,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8340180/v1/0295a90a-66ec-459f-817b-43eb3c196b29.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lutein and Zeaxanthin Provide Protection Against Aflatoxin B1-Induced Liver and Kidney Toxicity: Insights from in Silico and in Vivo Studies ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAflatoxin B\u003csub\u003e1\u003c/sub\u003e (AFB\u003csub\u003e1\u003c/sub\u003e) stands as the most potent aflatoxin produced by select \u003cem\u003eAspergillus species\u003c/em\u003e and poses a significant threat to food safety worldwide. Its frequent presence in crops such as maize and peanuts, especially in warm and humid regions [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], has led regulatory agencies\u0026mdash;including those in the European Union and United States\u0026mdash;to establish safety limits for human intake ranging from 2 to 30 \u0026micro;g/kg [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Nevertheless, despite regulatory efforts and multiple mitigation strategies, AFB\u003csub\u003e1\u003c/sub\u003e contamination remains persistent in food and feed, contributing to outbreaks of aflatoxicosis and a host of related diseases, including liver cancer, severe hepatitis, Reye's syndrome, gallbladder cancer, kwashiorkor, and respiratory illnesses [\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The toxicity of AFB\u003csub\u003e1\u003c/sub\u003e is strikingly broad, affecting organs such as the liver, kidneys, pancreas, brain, heart, and gonads in both humans and animals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The liver is particularly susceptible, serving as the primary site for AFB\u003csub\u003e1\u003c/sub\u003e metabolism via the cytochrome P450 enzyme system\u0026mdash;most notably CYP1A2 and CYP3A4. This process generates a highly reactive epoxide (AFB\u003csub\u003e1\u003c/sub\u003e-8,9-epoxide), which forms DNA adducts like AFB\u003csub\u003e1\u003c/sub\u003e-N7-guanine, resulting in genetic mutations and carcinogenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Mechanistic studies have revealed that AFB\u003csub\u003e1\u003c/sub\u003e induces toxicity through several interrelated pathways: oxidative stress, inflammation, and apoptosis [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Specifically, AFB\u003csub\u003e1\u003c/sub\u003e suppresses antioxidant defenses, stimulates the production of reactive oxygen species (ROS) and lipid peroxidation (by inhibiting the Nrf2/HO-1 pathway) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and promotes mitochondrial-mediated apoptosis by modulating apoptotic markers such as cytochrome c, caspase 3, caspase 8, and Bcl-2-associated X protein (Bax), while downregulating B-cell lymphoma 2 (Bcl-2) [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLutein (LUT) and zeaxanthin (ZEA), two naturally occurring xanthophyll carotenoids found abundantly in green leafy vegetables, corn, and egg yolks, have garnered attention for their health-promoting properties that extend beyond ocular benefits[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Recent research underscores their antioxidant, anti-inflammatory, and anti-apoptotic activities in models of neurotoxicity, hepatotoxicity, and nephrotoxicity [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. LUT and ZEA have been shown to reduce oxidative stress by scavenging ROS, preserve cellular membrane integrity, suppress pro-inflammatory cytokines (e.g., TNF-α, IL-6) via nuclear factor kappa-light-chain-enhancer of activated B cells NF-κB inhibition [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and modulate key molecular pathways including peroxisome proliferator-activated receptor gamma (PPARγ) and nuclear factor erythroid 2-related factor 2 (Nrf2). These effects help restore cellular homeostasis and limit tissue injury [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite mounting evidence of the protective effects of LUT and ZEA, their role in counteracting AFB1-induced hepatorenal toxicity remains insufficiently explored. This research hypothesizes that LUT and ZEA can attenuate AFB\u003csub\u003e1\u003c/sub\u003e-driven oxidative stress, inflammation, and apoptosis in hepatic and renal tissues. To test this, the study integrates molecular docking, network pharmacology, and in vivo analysis to investigate the mechanisms and potential target proteins involved in the protective actions of these carotenoids against AFB\u003csub\u003e1\u003c/sub\u003e toxicity. The insights gained from this work may guide the development of dietary strategies to mitigate the health risks posed by AFB\u003csub\u003e1\u003c/sub\u003e exposure, particularly in populations at elevated risk due to persistent contamination. In revealing the molecular underpinnings of LUT and ZEA\u0026rsquo;s protective effects, this study seeks to contribute meaningfully to the advancement of nutritional interventions and public health safeguards against aflatoxicosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNetwork Pharmacology\u003c/h2\u003e \u003cp\u003eThe 3D structure of Aflatoxin B\u003csub\u003e1\u003c/sub\u003e, Lutein, and Zeaxanthin were obtained through the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Potential targets of AFB\u003csub\u003e1\u003c/sub\u003e, Lutein and Zeaxanthin were predicted using the PharmMapper (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.lilabecust.cn/pharmmapper/\u003c/span\u003e\u003cspan address=\"https://www.lilabecust.cn/pharmmapper/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eand SwissTargetPrediction (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"https://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases assessed on 15th October, 2025. The SDF file of AFB\u003csub\u003e1\u003c/sub\u003e, Lutein and Zeaxanthin was uploaded to the PharmMapper, the dataset of human protein targets only (v2010, 2241) in PharmMapper, the default values for the rest parameters were kept, the job was submitted, and the screening results were awaited. Simultaneously, the SMILES from PubMed were uploaded to the SwissTargetPrediction database, and the results were saved as a CSV file. Subsequently, the targets collected from the PharmMapper and SwissTargetPrediction databases were filtered, and duplicates were removed using Microsoft Excel 2025 for subsequent analysis. The potential targets for hepatorenal toxicity were discovered using GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e assessed on 15th October, 2025, using \u0026ldquo;hepatorenal toxicity\u0026rdquo; as the keyword to recruit the targets of hepatorenal toxicity. Predicted results were exported, and the targets collected from the GeneCards database were filtered accordingly for subsequent analysis. Intersection targets of AFB\u003csub\u003e1\u003c/sub\u003e, LUT/ZEA, and Hepatorenal toxicity were obtained using the Venny 2.1 website. The PPI (protein\u0026ndash;protein interaction) network was collected by STRING 12.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), proteins were obtained on the STRING 12.0 database accessed on 17th October, 2025, and this given list of interactions was imported to predict the interaction of identified differentially expressed proteins (DEPs). The file of DEPs obtained from STRING was imported into Cytoscape (version 3.10.3) to construct the PPI network. The CytoHubba app plug-in in Cytoscape was used to compute the Maximum Clique Centrality (MCC) for identifying hub genes, thus creating a PPI network of the hub genes.\u003c/p\u003e \u003cp\u003eShiny G.O (0.85) accessed on 17th October, 2025, was used to perform gene ontology, a gene functional classification system that provides dynamically updated terms to comprehensively describe the properties and products of targets and the enrichment analysis of intersection targets was performed The Kyoto Encyclopedia of Genes and Genomes (KEGG) can systematically analyze the gene functions and metabolic terms pathways involved in the target gene functions and metabolic pathways involved in the target genes, which is helpful to fully understand the overall effect of the predicted targets in the organism.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Docking\u003c/h3\u003e\n\u003cp\u003eMolecular docking was performed to assess the binding affinity and potential interaction sites between aflatoxin B1, zeaxanthin, lutein, and the selected hub genes, to elucidate the possible inhibitory mechanisms. The 3D protein data bank (PDB) files of key targets (ALB, IL2, LGALS3, SRC, REN, EGFR, PRKACA, LCN2, F2, TTR) were retrieved from the RCSB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), assessed on 17th October, 2025. Molecular docking analysis using PyRx was conducted to predict the molecular interactions between all the genes, Aflatoxin B\u003csub\u003e1,\u003c/sub\u003e Lutein, and Zeaxanthin. Ligand preparation, protein preparation, binding sites recognition, docking, and visualization of docking modes were done using Discovery Studio 2021 software.\u003c/p\u003e\n\u003ch3\u003eChemical\u003c/h3\u003e\n\u003cp\u003eLutein \u0026amp; Zeaxanthin were purchased from Costco Wholesale Corporation, USA. Aflatoxin B\u003csub\u003e1\u003c/sub\u003e powder (AFB\u003csub\u003e1\u003c/sub\u003e, purity\u0026thinsp;\u0026ge;\u0026thinsp;98.0%, #1162-65-8, C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e, MW: 312.27g), high-density lipoprotein (HDL; Cat. No. MAK331), total cholesterol (TC; Cat. No. CS0005), and triglycerides (TG; Cat. No. MAK266) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Aspartate aminotransferase (AST; Cat. No. AS101), Alanine aminotransferase (ALT; Cat. No. AL146), creatinine (Cat. No. CR510), and urea (Cat. No. UR1068) assay kits were purchased from Randox Laboratories Limited (Crumlin, UK). Enzyme-linked Immunosorbent Assay kits for estimation of p53 (E-EL-H0910), Bcl-2 (E-EL-R0096), and BAX (E-EL-R0098) were purchased from E-labscience Biotechnology Co., Ltd, Wuhan, China. Monosodium phosphate (CAS No.: 7558\u0026ndash;80\u0026thinsp;\u0026minus;\u0026thinsp;7), disodium hydrogen phosphate (CAS No.: 7558\u0026ndash;79\u0026thinsp;\u0026minus;\u0026thinsp;4), sodium carbonate (CAS No.: 497\u0026ndash;19\u0026thinsp;\u0026minus;\u0026thinsp;8), sodium hydroxide (CAS No.: 1310\u0026ndash;73\u0026thinsp;\u0026minus;\u0026thinsp;2), sodium\u0026ndash;potassium tartrate (CAS No.: 6381\u0026ndash;59\u0026thinsp;\u0026minus;\u0026thinsp;5), and sodium chloride (CAS No.: 7647\u0026ndash;14\u0026thinsp;\u0026minus;\u0026thinsp;5) were obtained from BDH Ltd. (Poole, Dorset, UK) and William Hopkins Ltd. (Birmingham, UK). All other biochemical reagents and chemicals were obtained commercially and of analytical grade.\u003c/p\u003e\n\u003ch3\u003eAnimal care and welfare\u003c/h3\u003e\n\u003cp\u003eTen (10) weeks-old male Wistar rats (180\u0026ndash;230 g) were obtained from the Faculty of Veterinary Medicine, University of Ibadan College of Medicine (Ibadan, Nigeria). The experimental rats were acclimatised for 7 days before the commencement of the experimental period and housed in plastic cages with unrestricted access to standard feed (Breedwell\u0026reg; Feeds Limited, Ibadan, Nigeria) and freshwater. All rats were housed under standard laboratory conditions, with a natural photoperiod featuring a daily cycle of light and dark lasting approximately 12 hours at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. This experiment adheres to the 3Rs (replacement, reduction, and refining) guidelines for the use and care of experimental animals, approved by the University of Ibadan Animal Care and Use Research Ethics Committee (ACUREC No. UI-ACUREC/068\u0026ndash;0524/06) and conforms with the NIH publications volume 25, No.28 and revised in 1996 and EU Directive 2010/63/EU guidelines. Additionally, the animals' health was closely monitored, and appropriate measures were taken daily to ensure their maximum welfare, including checking fur condition, animal mobility, and body weight. Animals showing weight loss exceeding an acceptable limit, significant trauma, and inactivity for more than 24 hours were culled from the study.\u003c/p\u003e\n\u003ch3\u003eExperimental design and treatment\u003c/h3\u003e\n\u003cp\u003eTwenty-five (25) male Wistar rats were employed for the current study and were randomly assigned into five (5) cohorts of eight animals as follows:\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe control\u003c/b\u003e cohort received corn oil 2 mL/kg per os\u003c/p\u003e \u003cp\u003e \u003cb\u003eAflatoxin B\u003c/b\u003e \u003csub\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sub\u003e cohort (\u003cb\u003eAFB\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e)\u003c/b\u003e received aflatoxin B\u003csub\u003e1\u003c/sub\u003e (75 \u0026micro;g/kg per os)\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLutein/Zeaxanthin cohort\u003c/strong\u003e \u003cp\u003ereceived \u003cb\u003eLUT/ZEA (\u003c/b\u003e100 mg/kg per os)\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAFB\u003c/b\u003e \u003csub\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cb\u003eLUT/ZEA (low dose)\u003c/b\u003e: received 75\u0026micro;g/kg of \u003cb\u003eAFB\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e and 100 mg/kg of \u003cb\u003eLUT/ZEA\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eAFB\u003c/b\u003e \u003csub\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cb\u003eLUT/ZEA (high dose)\u003c/b\u003e: received 75\u0026micro;g/kg of \u003cb\u003eAFB\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e and 200 mg/kg of \u003cb\u003eLUT/ZEA\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eLUT/ZEA\u003c/b\u003e and \u003cb\u003eAFB\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e were dissolved in corn oil and administered orally for twenty-eight (28) days. The doses were selected based on previous literature: for AFB\u003csub\u003e1\u003c/sub\u003e [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] were used; for LUT/ZEA, references [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] were used. The experimental protocol and treatment adhered to these previously reported dosages documented in the scientific literature.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eOn day 29, the rats were weighed using a U.S. Solid Digital Analytical Balance (USS-DBS16, Cleveland, OH, USA), and blood samples were obtained from each rat via the retro-orbital sinus. The experimental rats were euthanised, and the liver and kidney tissue were removed and weighed to estimate the liver and kidney coefficient, mathematically expressed as a percentage ratio of tissue weight to body weight. The blood was allowed to clot, then centrifuged at 3000 g for 10 minutes to obtain serum samples, which were frozen at -20\u0026deg;C until kidney and liver function assays were performed. The liver and kidney tissues obtained were rinsed in cold 1.15% aqueous potassium chloride, homogenised (Teflon homogeniser) in phosphate buffer (0.1 M, pH 7.4), and centrifuged for 10 min at 15000 rpm to obtain supernatant for biochemical assay.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiochemical assay\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of serum enzyme biomarkers\u003c/h2\u003e \u003cp\u003eSerum samples were used to quantify creatinine, urea, high-density lipoprotein (HDL), total cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) using kits purchased from Randox Laboratories Limited (UK) according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein determination in the sample\u003c/h2\u003e \u003cp\u003eThe total protein content of the supernatants from liver and kidney homogenates was measured using the Lowry technique [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The tissue sample (7 \u0026micro;L) and distilled water (23 \u0026micro;L) were mixed in a microplate, followed by the addition of 150 \u0026micro;L of the alkaline CuSO₄ solution. The resulting mixture was allowed to settle at room temperature for ten minutes. After adding 15 \u0026micro;L of Folin-Ciocalteu solution, the mixture was incubated for 30 minutes at room temperature. Finally, the absorbance (750 nm) was acquired with a spectrophotometer against a reagent blank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the oxido-nitrosative stress\u003c/h2\u003e \u003cp\u003eThe level of superoxide dismutase (SOD) activity was measured by the inhibition of adrenaline auto-oxidation described by Misra and Fridovich [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Briefly, 5 \u0026micro;L of the sample and blank, along with 250 \u0026micro;L of 0.05 M carbonate buffer (pH 10.2), were added to a plate reader, followed by the addition of 30 \u0026micro;L of freshly prepared adrenaline. Absorbance was measured for 3 minutes at 480 nm, every 30 seconds. Catalase (CAT) activity was measured using Clairborne's technique [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], with hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) serving as the substrate. 25 \u0026micro;l of the sample and blank were mixed with 118 \u0026micro;l of a hydrogen peroxide solution (19 mM) in a plate reader, and the mixture was immediately read at 240 nm (1 min interval for 5 minutes). Reduced glutathione (GSH) was determined by applying the method of Jollow \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. After adding 80 \u0026micro;L of TCA, the mixture was combined with 80 \u0026micro;L of the sample and blank, vortexed, and then centrifuged at 4000 rpm for 5 minutes. Then, 50 \u0026micro;L of the TCA supernatant was combined with 150 \u0026micro;L of Ellman's reagent and incubated for 10 min at room temperature. The absorbance was then read at 412 nm using a plate reader. Glutathione-\u003cem\u003es\u003c/em\u003e-transferase (GST) activity was assessed using Habig's technique [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], which allowed the reaction to proceed for three minutes after the estimated medium was prepared. Readings were taken at 340 nm every 60 seconds and compared to the blank.\u003c/p\u003e \u003cp\u003eAlso, glutathione peroxidase (GPx) activity was biochemically assessed following the methods of Rotruck \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. A test tube was filled with 50 \u0026micro;L of phosphate buffer, 10 \u0026micro;L of NaN\u003csub\u003e3\u003c/sub\u003e, 20 \u0026micro;L of GSH, 10 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 50 \u0026micro;L of the sample, which was added last. After adding 50 \u0026micro;L of TCA and incubating for three minutes at 37˚C, the reaction mixture was centrifuged for five minutes at 3000 rpm. 100 \u0026micro;L of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e and 50 \u0026micro;L of DTNB were combined with 50 \u0026micro;L of the supernatant. Next, the absorbance was measured at 412 nm against a reagent blank that included 50 \u0026micro;L of DTNB, 100 \u0026micro;L of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, and 50 \u0026micro;L of distilled water.\u003c/p\u003e \u003cp\u003eThe total sulfhydryl cohort (TSH) was determined using Ellman's method [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. 150 \u0026micro;L of the sample, 100 \u0026micro;L of phosphate buffer, and 250 \u0026micro;L of distilled water were pipetted into an Eppendorf tube. The mixture was then left to stand. After pipetting 15 \u0026micro;L of Ellman's reagent into a microplate, 230 \u0026micro;L of the reaction mixture was added. The mixture was then left to stand for two minutes. At 412 nm, the absorbance was measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of an inflammatory biomarker\u003c/h2\u003e \u003cp\u003eA pro-inflammatory biomarker, Xanthine oxidase (XO), was assessed by measuring XO activity as a biomarker of inflammation. Quantification of XO was performed using the method described by Bergmeyer \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A 150-\u0026micro;L sample, 100 \u0026micro;L of phosphate buffer, and 250 \u0026micro;L of distilled water were pipetted into an Eppendorf tube. After that, the mixture was left to stand. After pipetting 15 \u0026micro;L of Ellman's reagent onto a microplate, 230 \u0026micro;L of the reaction mixture was added, and the mixture was left to stand for 2 minutes. At 412 nm, the absorbance was then measured.\u003c/p\u003e \u003cp\u003eMeanwhile, the protocols of Green \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] were utilised to estimate the amounts of nitric oxide (NO) for the NO assay. After the Griess reaction, the concentrations of nitrite in liver and kidney supernatants were determined by adding 100 \u0026micro;L of the samples to 100 \u0026micro;L of Griess reagent (0.1%) N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide in 5% phosphoric acid for 20 minutes at room temperature. The absorbance at 550 nm (OD 550) was measured and compared with a standard curve of sodium nitrite to estimate the nitrite concentration.\u003c/p\u003e \u003cp\u003eMyeloperoxidase (MPO) activity was measured by modifying the technique outlined by Trush et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. 200 \u0026micro;L of O-dianisidine dihydrochloride, 50 \u0026micro;L of diluted H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 7 \u0026micro;L of tissue homogenate were added to a 96-well plate and read at 460 nm for four minutes, at 30-second intervals, expressed as units of MPO/mg tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology and Histomorphometry Evaluation\u003c/h2\u003e \u003cp\u003eThe liver and Kidney tissues were excised immediately after sacrifice, rinsed in ice-cold saline and fixed in 10% formalin for 48 hours. Fixed tissues were dehydrated through graded ethanol (70\u0026ndash;100%), cleared in xylene, and embedded in paraffin wax. Serial sections of 4\u0026ndash;5 \u0026micro;m thickness were cut using a rotary microtome and mounted on glass slides. Sections were stained with hematoxylin and eosin (H\u0026amp;E) following standard protocols to visualize glomerular and tubular morphology\u003c/p\u003e \u003cp\u003eThe liver and kidney tissue (n\u0026thinsp;=\u0026thinsp;3/group) were excised immediately after sacrifice, rinsed in ice-cold saline and fined in 10% formalin for 48 hours. The fixed tissues were dehydrated in graded ethanol (70\u0026ndash;100%), cleared in xylene and embedded in paraffin wax. Microtome section (4\u0026ndash;5 \u0026micro;m) were obtained and mounted on glass slides and stained with hematoxylin and eosin (H\u0026amp;E) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. histoarchitecture of the liver and kidney section were determined using light microscope at 400X and ImageJ software was used to obtain average hepatocyte count and average diameter of the glomerulus in five non-overlapping photomicrographs. Semi-quantitative scoring scale of tubular and interstitial lesion was performed on five non-overlapping photomicrographs/kidney tissue slide including tubular necrosis, tubular degeneration, tubular dilatation, basement membrane thickening and interstitial fibrosis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data obtained were normalised using the D'Argostino-Pearson Omnibus and Shapiro-Wilk tests and expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The mean differences, analysed using one-way analysis of variance (ANOVA) followed by a post-hoc test (Tukey test), were analysed using GraphPad Prism (version 10) (CA, USA). The significance level was set at *\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNetwork Pharmacology\u003c/h2\u003e \u003cp\u003ePotential targets of AFB\u003csub\u003e1\u003c/sub\u003e, hepatorenal toxicity, and LUT/ZEA were identified using online databases as mentioned earlier. Approximately 346 targets of \u0026ldquo;AFB\u003csub\u003e1\u003c/sub\u003e,\u0026rdquo; 257 targets of \u0026ldquo;hepatorenal toxicity,\u0026rdquo; and 330 targets of LUT/ZEA were found. 13 intersection targets of AFB\u003csub\u003e1\u003c/sub\u003e, Hepatorenal Toxicity, and LUT/ZEA were collected for subsequent analysis. ALB, IL2, LGALS3, SRC, REN, EGFR, PRKACA, LCN2, F2, and TTR were the top 10 hub targets according to MCC values of AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatorenal toxicity mitigated by LUT/ZEA. The PPI network for AFB\u003csub\u003e1\u003c/sub\u003e and hepatorenal toxicity had 22 nodes and 77 edges, while AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatorenal toxicity mitigated by LUT/ZEA had 13 nodes and 30 edges, constructed using the intersection targets of AFB\u003csub\u003e1\u003c/sub\u003e, hepatorenal toxicity, and LUT/ZEA. The topological parameters of the PPI network were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In this study, there were 28 edges and 10 nodes in the PPI network of hub targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe enrichment analysis results were filtered using -log\u003csub\u003e10\u003c/sub\u003e (FDR), the top 20 terms of GO enrichment were selected and displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The findings demonstrated that the targets of AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatorenal toxicity mitigated by LUT/ZEA involved biological processes such as hormone transport, response to stress, regulation of multicellular organismal-level homeostasis, regulation of body fluid level, hormone secretion and tissue remodelling. The targets were related to the cellular component (CC) in extracellular membrane-bound organelles, cytoplasmic vesicles, dendritic filopodia, etc. The molecular function (MF) terms of targets included protein kinase activity, molecular function regulator activity, epidermal growth factor receptor activity, ATPase binding, hormone binding, etc. The top 20 terms of KEGG pathways enrichment were selected and displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG. The KEGG analysis showed that the targets of AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatorenal toxicity were mainly enriched in bladder cancer, thyroid hormone synthesis, chemical carcinogenesis receptor activity, oxytocin signaling pathways, estrogen signaling pathway, and pathways in cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eMolecular docking was applied to ALB, IL2, LGALS3, SRC, REN, EGFR, PRKACA, LCN2, F2, and TTR, and the docking interactions of these targets with AFB\u003csub\u003e1\u003c/sub\u003e, lutein, and zeaxanthin are presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. AFB\u003csub\u003e1\u003c/sub\u003e bound strongly to ALB through a carbon-hydrogen bond with Ala175, and pi-sigma interaction with Ala176. Lutein had a weak binding with ALB through alkyl interactions with Ala176, Lys519, Pro180, Lys436, Ala191, and Leu179. Zeaxanthin also bound weakly through polar interactions with Leu210, Leu264, and Val270 (Figs.\u0026nbsp;5A1, 5A2, 5A3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAFB\u003csub\u003e1\u003c/sub\u003e bound strongly to IL2 through conventional hydrogen bond with Arg120, carbon-hydrogen bonds with Glu116 and Thr111, and pi-sigma interaction with Met46. Lutein doesn\u0026rsquo;t bind strongly to ALB but forms an alkyl, pi-alkyl bond with His16, Ile122. Zeaxanthin had an unfavorable bump with Asn26, which shows a repulsion between zeaxanthin and the binding site of IL2 (Fig.\u0026nbsp;5B1-5B\u003cb\u003e3\u003c/b\u003e). AFB\u003csub\u003e1\u003c/sub\u003e bound strongly to IL2 through conventional hydrogen bond with Arg120, carbon-hydrogen bond with Glu116 and Thr111, and pi-sigma interaction with Met46. Lutein doesn\u0026rsquo;t bind strongly to ALB but forms an alkyl, pi-alkyl bond with His16, Ile122. Zeaxanthin had an unfavorable bump with Asn26, which shows a repulsion between zeaxanthin and the binding site of IL2 (Fig.\u0026nbsp;5B1- 5B\u003cb\u003e3\u003c/b\u003e). AFB\u003csub\u003e1\u003c/sub\u003e bound strongly to LGALS3 through conventional hydrogen interactions with HIS158 and Lys176, carbon-hydrogen interactions with Gly182 and Asn180, and pi-pi stacked interaction with Trp181. Lutein binds strongly to LGALS3 through conventional hydrogen interaction with Asn160, and pi-sigma interaction with Trp181. Zeaxanthin binds to LGALS3 through pi-sigma interactions with Trp181 (Fig.\u0026nbsp;6A1-6A\u003cb\u003e3\u003c/b\u003e). AFB\u003csub\u003e1\u003c/sub\u003e interacted with SRC through carbon carbon-hydrogen bond with Tyr479, a carbon-hydrogen bond with VAL461, and a pi-sulfur bond with Met481. Lutein interacted with SRC through pi-sigma bond with His492, unfavourable bonds with Glu505, Trp499, Met481, and Cys496. Zeaxanthin interacted with SRC through a conventional hydrogen bond with Glu505, and unfavourable bonds with Met481, Cys496, Thr453, and His492 (Fig.\u0026nbsp;6B1-6B\u003cb\u003e3\u003c/b\u003e). AFB\u003csub\u003e1\u003c/sub\u003e interacted with REN through a conventional hydrogen bond with Gly96 and Gly95, a carbon-hydrogen bond with Asn184 and Phe331, and a pi-pi T-shaped bond with Phe318. Lutein interacted with REN through a conventional hydrogen bond with Ser161 and Lys270. Zeaxanthin interacted with REN through a conventional hydrogen bond with Asp160 and Glu162, and an unfavourable bump bond with Tyr285 (Fig.\u0026nbsp;7A1-7A\u003cb\u003e3\u003c/b\u003e). AFB\u003csub\u003e1\u003c/sub\u003e interacted with EGFR through a conventional hydrogen bond with His209. Lutein interacted weakly with EGFR through alkyl bonds. Zeaxanthin interacted weakly with EGFR through an alkyl bond (Fig.\u0026nbsp;7B1-7B\u003cb\u003e3)\u003c/b\u003e. AFB\u003csub\u003e1\u003c/sub\u003e interacted with PRKACA through conventional hydrogen bond with Arg133, carbon-hydrogen bond with Pro236, pi-cation and pi-anion with Glu203 and Phe129. Lutein interacted with PRKACA through pi-sigma bond with Phe129 and alkyl bond with Ala240, Phe239, Phe240, Ala240 and Lys168. Zeaxanthin interacted with PRKACA through pi-sigma bond with Phe129, alkyl and pi-alkyl bond with Ala240, Phe239, Phe129, Lys168 (Fig.\u0026nbsp;8A1-8A\u003cb\u003e3\u003c/b\u003e). AFB\u003csub\u003e1\u003c/sub\u003e interacted with LCN2 through conventional hydrogen bonds with Tyr126, Ser88, Arg101, and pi-cation bonds with Lys154. Lutein interacted with LCN2 through a conventional hydrogen bond with Asp67 and Glu64, and pi-alkyl bond with Trp99 and Tyr120. Zeaxanthin interacted with LCN2 through carbon carbon-hydrogen bond with Pro68, and unfavourable bumps with Leu62, Gln69, and Val187 (Fig.\u0026nbsp;8B1-8B\u003cb\u003e3\u003c/b\u003e). AFB\u003csub\u003e1\u003c/sub\u003e interacted with F2 through a conventional hydrogen bond with Arg221 and Arg221 and pi-carbon bond with Arg173. His209. Lutein interacted with F2 through a conventional hydrogen bond with Gly186. Zeaxanthin interacted weakly with F2 through alkyl bonds (Fig.\u0026nbsp;9A1-9A\u003cb\u003e3\u003c/b\u003e). AFB\u003csub\u003e1\u003c/sub\u003e interacted with TTR through conventional hydrogen bond with Lys35, Trp41, and Lys70, carbon-hydrogen bond with Glu89 and His90, and pi-anion bond with Glu72. Lutein interacted with TTR through van der Waals bonds with His31, Asn74, Glu72, Lys70, and Ala29. Zeaxanthin interacted with TTR through a pi-alkyl bond with Trp41(Fig.\u0026nbsp;9B1-9B\u003cb\u003e3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Lutein/Zeaxanthin on Relative Organ Weight in AFB\u003c/b\u003e \u003csub\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eTreated Rats.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effect of \u003cb\u003eLutein/Zeaxanthin\u003c/b\u003e \u003cb\u003e(\u003c/b\u003eLUT/ZEA) on the hepatorenal-somatic index of rats exposed to AFB\u003csub\u003e1\u003c/sub\u003e is depicted in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. At the end of the experimental period, rats exposed to AFB\u003csub\u003e1\u003c/sub\u003e had higher (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) relative liver and kidney weights compared to the control cohort. In contrast, the AFB\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;LUT/ZEA\u003csub\u003e2\u003c/sub\u003e cohort (high dose; 200 mg/kg) showed increased body weight gain, whereas the low-dose cohort (100 mg/kg) did not, compared to the AFB\u003csub\u003e1\u003c/sub\u003e-only administered cohort.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of the administration of Lutein/Zeaxanthin in Rats Treated Aflatoxin B\u003csub\u003e1\u003c/sub\u003e induced toxicity on the body weight, organ weight, and relative organ weight of rats\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeights(g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAFB\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLut/Zea\u003c/p\u003e \u003cp\u003e(100 mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAFB\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Lut/Zea\u003c/p\u003e \u003cp\u003e(100 mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAFB\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Lut/Zea\u003c/p\u003e \u003cp\u003e(200 mg/kg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal Body weight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e216.1\u0026thinsp;\u0026plusmn;\u0026thinsp;32.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e204.8\u0026thinsp;\u0026plusmn;\u0026thinsp;18.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e215.4\u0026thinsp;\u0026plusmn;\u0026thinsp;20.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e211.1\u0026thinsp;\u0026plusmn;\u0026thinsp;16.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e216.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial Body Weight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e213.7\u0026thinsp;\u0026plusmn;\u0026thinsp;25.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e201.1\u0026thinsp;\u0026plusmn;\u0026thinsp;17.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e209.8\u0026thinsp;\u0026plusmn;\u0026thinsp;18.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e206.8\u0026thinsp;\u0026plusmn;\u0026thinsp;16.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e213.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody Weight Gain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;14.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.63\u0026thinsp;\u0026plusmn;\u0026thinsp;4.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.625\u0026thinsp;\u0026plusmn;\u0026thinsp;5.181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.38\u0026thinsp;\u0026plusmn;\u0026thinsp;6.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3.29\u0026thinsp;\u0026plusmn;\u0026thinsp;6.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRelative Liver weight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.321\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.247\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.133\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRelative Kidney weight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eRats were administered with Aflatoxin B\u003csub\u003e1\u003c/sub\u003e (75\u0026micro;g/kg BW), Lutein/Zeaxanthin (100 mg/kg BW), Aflatoxin B\u003csub\u003e1\u003c/sub\u003e (75\u0026micro;g/kg per BW) plus Lutein/Zeaxanthin (100 and 200 mg/kg per BW), or vehicle for 28 consecutive days of feeding (n\u0026thinsp;=\u0026thinsp;8 per group). Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Differences between the treatment groups were analyzed by student's t-test. AFB\u003csub\u003e1\u003c/sub\u003e: Aflatoxin B\u003csub\u003e1\u003c/sub\u003e; LUT/ZEA: Lutein/Zeaxanthin.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDocking scores of the target genes with AFB\u003csub\u003e1,\u003c/sub\u003e Lutein, and Zeaxanthin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget Genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePDB ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eDocking Scores (Kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eAFB\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eLUT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eZEA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1AO6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1M47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLGALS3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3T1M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-9.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSRC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1FMK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eREN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2I4Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-8.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEGFR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4R3P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-6.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePRKACA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7Y1G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-8.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-5.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLCN2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3BX8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-7.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2AFQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1DVQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-5.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of the administration of Lutein/Zeaxanthin on semi-quantitative scoring of tubular and interstitial lesion in rats exposed to Aflatoxin B\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSemi-quantitative scoring scale\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAFB\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(75\u0026micro;g/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLut/Zea\u003c/p\u003e \u003cp\u003e(100 mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAFB\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Lut/Zea\u003c/p\u003e \u003cp\u003e(100 mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAFB\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Lut/Zea\u003c/p\u003e \u003cp\u003e(200 mg/kg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubular necrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubular Degeneration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-/+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-/+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubular Dilatation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-/+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasement membrane thickening\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-/+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-/+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-/+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInterstitial fibrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eRats were administered with Aflatoxin B\u003csub\u003e1\u003c/sub\u003e (75\u0026micro;g/kg BW), Lutein/Zeaxanthin (100 mg/kg BW), Aflatoxin B\u003csub\u003e1\u003c/sub\u003e (75\u0026micro;g/kg per BW) plus Lutein/Zeaxanthin (100 and 200 mg/kg per BW), or vehicle for 28 consecutive days of feeding (n\u0026thinsp;=\u0026thinsp;3/ group). Semi-quantitative scoring scale is indicated by absence (-) or presence (+) of tubular or interstitial lesion. AFB\u003csub\u003e1\u003c/sub\u003e: Aflatoxin B\u003csub\u003e1\u003c/sub\u003e; LUT/ZEA: Lutein/Zeaxanthin.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffects of LUT/ZEA on Liver and Kidney Function Biomarkers in AFB\u003csub\u003e1\u003c/sub\u003e Treated Rats\u003c/h2\u003e \u003cp\u003eThe impact of co-exposure to AFB\u003csub\u003e1\u003c/sub\u003e and LUT/ZEA on the serum indicators of hepatic function is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. Compared to the control cohort, AFB\u003csub\u003e1\u003c/sub\u003e-treatment significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) increased serum AST [F (4, 15)\u0026thinsp;=\u0026thinsp;35.55, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] and ALT [F (4, 15)\u0026thinsp;=\u0026thinsp;12.58, P\u0026thinsp;=\u0026thinsp;0.0001] activity, creatinine [F (4, 15)\u0026thinsp;=\u0026thinsp;27.31, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] and urea [F (4, 15)\u0026thinsp;=\u0026thinsp;11.92, P\u0026thinsp;=\u0026thinsp;0.0001] level. Conversely, treatment with AFB\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;LUT/ZEA (100 mg/kg and 200 mg/kg) caused a significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) reduction in AST and ALT activity, creatinine, and urea levels when compared to the AFB\u003csub\u003e1\u003c/sub\u003e-treated cohort.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEffects of LUT/ZEA on Lipid Profile in AFB\u003csub\u003e1\u003c/sub\u003e Treated Rats\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e outlines the effect of the co-exposure to AFB\u003csub\u003e1\u003c/sub\u003e and LUT/ZEA on lipid profile. Compared to the control cohort, the AFB\u003csub\u003e1\u003c/sub\u003e-treated cohort significantly increased total cholesterol (TC) [F (4, 15)\u0026thinsp;=\u0026thinsp;15.25, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001], triglyceride [F (4, 15)\u0026thinsp;=\u0026thinsp;18.52, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] and decreased high-density lipoprotein level [F (4, 15)\u0026thinsp;=\u0026thinsp;18.74, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001]. At the same time, AFB\u003csub\u003e1\u003c/sub\u003e was co-treated with different doses of LUT/ZEA (100 mg/kg and 200 mg/kg), resulting in significantly lower TC and TG levels and increased HDL levels, compared to the AFB\u003csub\u003e1\u003c/sub\u003e-treated cohort.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Lutein/Zeaxanthin on the Hepatorenal Antioxidant System in AFB\u003csub\u003e1\u003c/sub\u003e Treated Rats\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e illustrates the impact of lutein and zeaxanthin on liver and kidney antioxidant biomarkers of rats exposed to 75 \u0026micro;g/mg AFB\u003csub\u003e1\u003c/sub\u003e. Experimental rats treated with AFB\u003csub\u003e1\u003c/sub\u003e alone showed significantly reduction (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) in the liver (upper panel) and the kidney (lower panel) SOD [F (4, 15)\u0026thinsp;=\u0026thinsp;26.04, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F (4, 15)\u0026thinsp;=\u0026thinsp;67.54, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001], CAT [F (4, 15)\u0026thinsp;=\u0026thinsp;20.02, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001], GPx [F (4, 15)\u0026thinsp;=\u0026thinsp;35.23, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F (4, 15)\u0026thinsp;=\u0026thinsp;84.00, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001], GST [F (4, 15)\u0026thinsp;=\u0026thinsp;23.98P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F (4, 15)\u0026thinsp;=\u0026thinsp;29.09, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F (4, 15)\u0026thinsp;=\u0026thinsp;12.22, P\u0026thinsp;=\u0026thinsp;0.0001, GSH [F (4, 15)\u0026thinsp;=\u0026thinsp;25.08, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F (4, 15)\u0026thinsp;=\u0026thinsp;12.22, P\u0026thinsp;=\u0026thinsp;0.0001], and TSH [F (4, 15)\u0026thinsp;=\u0026thinsp;9.732, P\u0026thinsp;=\u0026thinsp;0.0004; F (4, 15)\u0026thinsp;=\u0026thinsp;21.81, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] levels compared to the control cohort. On the other hand, in the administration of LUT/ZEA at doses of 100 mg/kg and 200 mg/kg, there was a significant increase in the antioxidant enzymatic activities. Specifically, there was an increase (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) in liver and kidney SOD and CAT activities at both doses, 100 mg/kg and 200 mg/kg.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Lutein/Zeaxanthin on the hepatorenal oxido-inflammatory responses in AFB1-treated rats\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e presents the effect of LUT/ZEA treatment on the mediators of oxidative stress and inflammatory response assessed in the liver (upper panel) and kidney (lower panel) of rats exposed to AFB\u003csub\u003e1\u003c/sub\u003e. Compared to the control cohort, the AFB\u003csub\u003e1\u003c/sub\u003e-treated cohort significantly increased hepatorenal XO [F (4, 15)\u0026thinsp;=\u0026thinsp;24.52, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F (4, 15)\u0026thinsp;=\u0026thinsp;28.94, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001], NO [F (4, 15)\u0026thinsp;=\u0026thinsp;18.04, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F (4, 15)\u0026thinsp;=\u0026thinsp;33.95, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001], and MPO [F (4, 15)\u0026thinsp;=\u0026thinsp;28.36, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F (4, 15)\u0026thinsp;=\u0026thinsp;25.82, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] activity compared to the control. On the other hand, AFB\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;LUT/ZEA (100 mg/kg and 200 mg/kg) significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) decreased liver and kidney XO, NO, and MPO activities at 100 mg/kg, and a significant (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) decrease in these activities at 200 mg/kg.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEffect of Lutein/Zeaxanthin on the hepatorenal tumor and apoptotic biomarkers in AFB\u003csub\u003e1\u003c/sub\u003e-treated rats\u003c/h2\u003e \u003cp\u003eApoptosis biomarkers were further assessed to investigate the impact of LUT/ZEA in rats exposed to AFB\u003csub\u003e1\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Rats exposed to AFB\u003csub\u003e1\u003c/sub\u003e had higher TP53 [F (4, 10)\u0026thinsp;=\u0026thinsp;56.12, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, F (4, 10)\u0026thinsp;=\u0026thinsp;15.36, P\u0026thinsp;=\u0026thinsp;0.0003], BAX [F (4, 10)\u0026thinsp;=\u0026thinsp;20.63, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F (4, 10)\u0026thinsp;=\u0026thinsp;16.25, P\u0026thinsp;=\u0026thinsp;0.0002] and reduced Bcl2 [F (4, 10)\u0026thinsp;=\u0026thinsp;13.43; F (4, 10)\u0026thinsp;=\u0026thinsp;7.291, P\u0026thinsp;=\u0026thinsp;0.0051] concentrations and an increase in BAX/Bcl2 ratio [F (4, 10)\u0026thinsp;=\u0026thinsp;82.29, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F (4, 10)\u0026thinsp;=\u0026thinsp;20.40, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] than control rats. However, rats co-treated with AFB\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;LUT/ZEA-treated rats demonstrated a substantial decrease in TP53, BAX, and reduced Bcl2 level, which increased compared to AFB\u003csub\u003e1\u003c/sub\u003e-only treated rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Lutein/Zeaxanthin on the hepatorenal histological architecture in AFB\u003csub\u003e1\u003c/sub\u003e-treated rats\u003c/h2\u003e \u003cp\u003eH\u0026amp;E microscopic examination of the liver and kidney are illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e16\u003c/span\u003e and \u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e17\u003c/span\u003e. In the liver section, control and LUT/ZEA only treated rats reveal normal histopathology indicated by significant increase in viable hepatocyte count. AFB\u003csub\u003e1\u003c/sub\u003e-only treated rats demonstrated decrease in hepatocyte count [F (4, 10)\u0026thinsp;=\u0026thinsp;31.13, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001], inflammatory cell infiltration, necrosis. AFB\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;LUT/ZEA-treated rats confirmed alleviation specifically at dose of 200 mg/kg compered to AFB\u003csub\u003e1\u003c/sub\u003e-only treated rats. in the kidney, control and LUT/ZEA only treated rats revealed normal glomeruli, the absences of tubular necrosis, tubular degeneration, tubular dilatation, basement membrane thickening and interstitial fibrosis. Experimental rat treated with AFB\u003csub\u003e1\u003c/sub\u003e-only showed reduction in glomeruli [F (4, 10)\u0026thinsp;=\u0026thinsp;21.35, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001], obliterated capillary structure and inflammatory cell infiltration and notable tubular necrosis, tubular degeneration, tubular dilatation, basement membrane thickening and interstitial fibrosis. AFB\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;LUT/ZEA-treated rats at 100 and 200 mg/kg reversed glomeruli and tubular lesion caused by AFB\u003csub\u003e1\u003c/sub\u003e-only treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides an integrated assessment of the protective potential of lutein and zeaxanthin (LUT/ZEA) against Aflatoxin B\u003csub\u003e1\u003c/sub\u003e (AFB\u003csub\u003e1\u003c/sub\u003e)-induced hepatorenal toxicity, combining network pharmacology, molecular docking, and \u003cem\u003ein vivo\u003c/em\u003e validation. The findings contribute valuable insights into the molecular mechanisms underlying LUT/ZEA\u0026rsquo;s mitigating effects, highlighting their promise as multi-targeted agents for alleviating mycotoxin-induced organ injury. AFB\u003csub\u003e1\u003c/sub\u003e is recognized as a potent hepatotoxin and nephrotoxin, with a well-documented ability to induce oxidative stress, inflammation, apoptosis, and mitochondrial dysfunction [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and likely deleterious mutations in sites where AFB\u003csub\u003e1\u003c/sub\u003e causes DNA damage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], These processes culminate in cellular damage and increase the risk of carcinogenesis, as supported by previous literature and reinforced by the current results.. Despite increasing knowledge of AFB\u003csub\u003e1\u003c/sub\u003e\u0026rsquo;s multi-organ toxicity, therapeutic interventions remain limited, underscoring the need for novel protective strategies.\u003c/p\u003e \u003cp\u003eRecent epidemiological and experimental evidence suggests that higher dietary intakes of carotenoids are associated with a reduced risk of liver disease, particularly non-alcoholic fatty liver disease (NAFLD)[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Lutein and zeaxanthin, notable for their antioxidant, anti-inflammatory, and anti-apoptotic properties, have demonstrated organ-protective effects in various tissues, including the eye, brain, heart, skin, and liver.\u003c/p\u003e \u003cp\u003eNetwork pharmacology analyses offered further mechanistic understanding by identifying a set of central hub genes (ALB, EGFR, IL2, REN, LCN2, SRC, LGALS3, PRKACA, F2, TTR) involved in AFB1-related hepatorenal toxicity. The enrichment of molecular functions related to epidermal growth factor receptor activity and kinase signaling, particularly involving EGFR and SRC, connects these targets to cellular proliferation, survival, and stress responses. Moreover, involvement of interleukin-2 (IL-2) receptor binding points to the role of LUT/ZEA in modulating immune and inflammatory cascades, although the lower binding affinity for IL-2 compared to AFB\u003csub\u003e1\u003c/sub\u003e suggests limited efficacy in suppressing certain inflammatory pathways.\u003c/p\u003e \u003cp\u003eGO biological process enrichment underscored the negative regulation of mitochondrial depolarization as a core protective mechanism. Since mitochondrial dysfunction is central to AFB1-induced cytotoxicity, the ability of LUT/ZEA to stabilize cellular bioenergetics reflects a crucial aspect of their protective profile. KEGG pathway analyses further highlighted the relevance of EGFR tyrosine kinase inhibitor resistance, ErbB signaling, and renin secretion pathways\u0026mdash;linking these findings directly to mechanisms of chronic renal injury and carcinogenesis.\u003c/p\u003e \u003cp\u003eMolecular docking simulations revealed that LUT/ZEA exhibited strong binding affinities for several key hub proteins, notably REN and LGALS3, surpassing AFB\u003csub\u003e1\u003c/sub\u003e in binding strength for these targets. This implies that LUT/ZEA may function as effective inhibitors of pro-inflammatory, proliferative, and fibrotic pathways. The ability of zeaxanthin and lutein to bind more effectively to LGALS3, a principal mediator of fibrosis, and REN, a regulator of the renin-angiotensin-aldosterone system, provides a molecular basis for their observed systemic protective effects. LUT/ZEA\u0026rsquo;s interactions with LCN2 and F2 further support their role in attenuating acute kidney injury and hepatic complications.\u003c/p\u003e \u003cp\u003eThe present study demonstrated that LUT/ZEA administration in a rat model exposed to AFB\u003csub\u003e1\u003c/sub\u003e resulted in notable reductions in biochemical markers indicative of liver damage (AST and ALT, which play roles in amino acid metabolism and are released when the liver is damaged) - and kidney (urea and creatine) injury [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Improvements in serum levels of ALT, AST, creatinine, and urea indicate that LUT/ZEA effectively mitigate AFB\u003csub\u003e1\u003c/sub\u003e-induced tissue injury. These results support earlier findings [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and align with the rise observed in the hepatorenal somatic index, reflecting injury following AFB\u003csub\u003e1\u003c/sub\u003e toxicity. However, rats treated with LUT/ZEA exhibited reversed effects, demonstrated by lower serum levels of AST, ALT, creatinine, and urea, implying a protective role against liver and kidney damage. Notably, the marked reduction in enzyme activity and metabolic waste in the bloodstream suggests this protective effect against AFB\u003csub\u003e1\u003c/sub\u003e compromised liver and kidneys structural integrity. This biochemical improvement is corroborated by histopathological findings, which demonstrates that LUT/ZEA treatment attenuated hepatocyte necrosis, inflammatory cell infiltration as well as glomeruli and tubular degeneration and interstitial fibrosis. The liver is essential for energy and lipid metabolism, and signs of liver damage are often linked to lipid buildup [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The present results indicate that AFB\u003csub\u003e1\u003c/sub\u003e disrupts lipid metabolism, as evidenced by higher triglyceride (TG) and total cholesterol (TC) levels, along with reduced high-density lipoprotein (HDL)\u0026mdash;known as good cholesterol\u0026mdash;signalling dyslipidemia, a common marker of hepatic parenchymal cell harm [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Furthermore, LUT/ZEA supplementation enhanced lipid profiles, reducing triglycerides and total cholesterol while increasing high-density lipoprotein levels, thereby counteracting dyslipidemia, a common consequence of hepatic damage. This observation supports prior research suggesting LUT/ZEA as an effective intervention for dyslipidemia, especially in older adults [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAFB\u003csub\u003e1\u003c/sub\u003e-related hepatorenal injury results in an overproduction of free radicals and oxidative stress, which plays a key role in AFB\u003csub\u003e1\u003c/sub\u003e potent carcinogenicity [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. These free radicals can harm cell membranes by damaging biomolecules such as proteins, lipid components, and DNA, leading to lipid peroxidation, reduced endogenous antioxidant activity, and a loss of cellular function and structural integrity [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. However, supplementation with LUT/ZEA has been associated with a reduction in oxidative stress [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This protective effect is connected to increased antioxidant activity, including superoxide dismutase (SOD), catalase (CAT), the glutathione system (GPx, GSH, GST), and total sulfhydryl (TSH)\u0026mdash;which neutralise harmful reactive oxygen species, like superoxide radicals and hydrogen peroxide (H₂O₂), converting them into harmless substances such as water and oxygen via the Fenton reaction. Evidence shows that AFB\u003csub\u003e1\u003c/sub\u003e causes oxidative stress in liver and kidney cells by accumulating free radicals, contributing to lipid peroxidation and tissue damage [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Our findings indicate that LUT/ZEA boosts hepatic and renal antioxidant activities, shown by higher levels of SOD, CAT, GPx, GSH, and GST, aligning with previous research [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] and supporting their role in protecting cell membranes from oxidative injury in the liver and kidneys [\u003cspan additionalcitationids=\"CR59 CR60\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Additionally, rats cotreated with LUT/ZEA displayed xanthine oxidase (XO) inhibitory activity, likely due to their molecular structure\u0026rsquo;s rich in conjugated double bonds, enabling effective scavenging of free radicals and inhibition of oxidative enzymes like XO [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Our results further demonstrated that administration of LUT/ZEA at doses of 100 and 200 mg/kg led to a reduction in inflammatory mediators, as indicated by decreased nitric oxide (NO) levels and myeloperoxidase (MPO) activity, both of which were significantly increased in rats exposed to AFB\u003csub\u003e1\u003c/sub\u003e. This suggests that LUT/ZEA may confer protection against NO production, thereby limiting the formation of peroxynitrite, a highly reactive and deleterious oxidant [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Additionally, the observed decrease in neutrophil count may have mitigated cell destruction, a pivotal component in the inflammatory and immune response to tissue injury [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Considering the ameliorative effects of LUT/ZEA on AFB\u003csub\u003e1\u003c/sub\u003e-induced inflammation and oxidative damage, we extended our investigation to examine their influence on apoptotic biomarker modulation following AFB\u003csub\u003e1\u003c/sub\u003e exposure.\u003c/p\u003e \u003cp\u003eApoptosis, a key contributor to liver and kidney injury, [\u003cspan additionalcitationids=\"CR66 CR67 CR68\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. AFB\u003csub\u003e1\u003c/sub\u003e has been shown to induce hepatic [\u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] and renal [\u003cspan additionalcitationids=\"CR74\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e] apoptosis by downregulating anti-apoptotic protein Bcl2 and upregulating pro-apoptotic proteins BAX and TP53. These biomarkers are indicative of the activation of multiple apoptotic pathways, including cytochrome c release and the subsequent activation of caspases 3 and 9 through mitochondrial signalling [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Apoptosis was also ameliorated by LUT/ZEA supplementation. The suppression of pro-apoptotic markers and the preservation of anti-apoptotic proteins suggest that LUT/ZEA help maintain the integrity and function of hepatic and renal tissues in the face of AFB\u003csub\u003e1\u003c/sub\u003e challenge.\u003c/p\u003e \u003cp\u003eTaken together, these findings establish LUT/ZEA as promising candidates for the prevention or amelioration of AFB\u003csub\u003e1\u003c/sub\u003e-induced hepatorenal toxicity. Their multifaceted mechanisms\u0026mdash;encompassing modulation of oxidative stress, inflammation, apoptosis, and key cellular pathways\u0026mdash;make them attractive for further investigation as dietary or therapeutic interventions, particularly in populations at risk for chronic mycotoxin exposure.\u003c/p\u003e \u003cp\u003eNevertheless, the study\u0026rsquo;s reliance on histopathological and biochemical endpoints indicates a need for broader molecular investigations. Future research should explore detailed signaling networks and validate findings in human subjects to optimize dosing and assess long-term safety.\u003c/p\u003e \u003cp\u003eIn conclusion, this study advances our understanding of the protective roles of lutein and zeaxanthin in counteracting the toxic effects of AFB\u003csub\u003e1\u003c/sub\u003e on liver and kidney tissues. The integration of network pharmacology, molecular docking, and in vivo experimentation provides a robust framework for elucidating their therapeutic potential and supports the ongoing pursuit of effective interventions against mycotoxin-induced organ damage.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFDR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFalse Discovery Rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLUT/ZEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLutein/Zeaxanthin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAFB\u003csub\u003e1\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAflatoxin B\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRAAS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRenin-Angiotensin-Aldosterone System\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlbumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDALS3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGalectin 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSRC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProto-oncogene, non-receptor tyrosine kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eREN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRenin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEGFR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpidermal Growth Factor Receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePRKACA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecAMP-dependent protein kinase catalytic subunit alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLCN2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipocalin 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eF2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCoagulation factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTTR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransthyretin gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the following students for their technical support: Japheth Auta, Praise Dyap, Marvellous Salami, Mark Nnamdi, Dooshima Bagu, Precious Taiye, Abisola Ibukunle, and Oluwadunsin Adekunle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocols for the care and use of experimental animals in this study were approved by the University of Ibadan, Animal Care and Use in Research Ethical Committee, with approval number:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors participated in the design, interpretation, and analysis of the study's data. SO conceptualised the study; SO, JAO, JC, JCN and OO conducted the research and analysed the preliminary data. SO supervised the investigation, and SO, JAO, JOB, VOE, HA, NHS, OO \u0026nbsp;and OO Proof checked the data for errors. EMP, SAA, IOA, VOE, HA, NHS and OO conducted the *in silico* study. SO, JC, \u0026nbsp; JAO, JOB, IOA, VOE, HA, EMP, NHS, OO and OO wrote and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the author(s).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors privately funded this research through their contributions and received no external grants from funding agencies in the commercial, not-for-profit, or public sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGuengerich FP et al (1996) Involvement of cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. 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Cell Death Dis 11(1):6\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"mycotoxin-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myre","sideBox":"Learn more about [Mycotoxin Research](http://link.springer.com/journal/12549)","snPcode":"12550","submissionUrl":"https://submission.nature.com/new-submission/12550/3","title":"Mycotoxin Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Network pharmacology, Aflatoxin B1, hepatorenal toxicity, lutein and zeaxanthin, oxido-inflammatory and DNA damage, apoptosis and molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-8340180/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8340180/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExposure to aflatoxin B\u003csub\u003e1\u003c/sub\u003e (AFB\u003csub\u003e1\u003c/sub\u003e) poses significant threats to food safety, increases food insecurity, and endangers public health due to its pronounced organ toxicity and carcinogenicity, especially affecting the hepatorenal system. This study integrates network pharmacology and molecular docking analyses to investigate the protective roles of the natural carotenoids lutein (LUT) and zeaxanthin (ZEA) against AFB\u003csub\u003e1\u003c/sub\u003e-induced toxicity. Key hub genes-ALB, IL2, LGALS3, SRC, REN, EGFR, PRKACA, LCN2, F2, and TTR- implicated in carcinogenesis, stress response, cellular homeostasis, and tissue remodelling were identified via Network Pharmacology analysis. Molecular docking demonstrated that LUT (-9.0 kcal/mol) and ZEA (-9.5 kcal/mol) have higher affinity to inflammation-related proteins (LGALS3) than AFB1 (-8.4 kcal/mol), suggesting their strong anti-fibrotic and antioxidant potentials. Lutein also showed the highest binding affinity for REN (-9.5 kcal/mol) compared to AFB\u003csub\u003e1\u003c/sub\u003e (-8.8 kcal/mol), suggesting a specific mechanism through which LUT may influence the RAAS pathway to reduce AFB\u003csub\u003e1\u003c/sub\u003e-induced kidney damage.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e experiments using Wistar rats (N\u0026thinsp;=\u0026thinsp;25; 10 weeks old, weighing 220\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) randomly assigned to five groups and treated as follows: Control group (corn oil only, 2 mL/kg), AFB1 only (75 \u0026micro;g/kg), LUT/ZEA only (100 mg/kg), AFB1 (75 \u0026micro;g/kg) plus LUT/ZEA1 (100 mg/kg), and LUT/ZEA2 (200 mg/kg) \u003cem\u003eper os\u003c/em\u003e for 28 days. And followed by the assessments of liver and kidney function, serum lipid profiles, enzymatic and non-enzymatic antioxidant levels, oxidative stress markers, inflammatory mediators, DNA damage, and apoptosis biomarkers were conducted using spectrophotometric methods, confirming that LUT/ZEA administration alleviated AFB\u003csub\u003e1\u003c/sub\u003e-induced liver and kidney damage, reduced oxidative stress, inflammation, DNA damage, and apoptosis in a dose-dependent manner. Histopathological analyses further validated these protective effects. Overall, the study highlights the potential of LUT and ZEA as natural agents for mitigating AFB\u003csub\u003e1\u003c/sub\u003e toxicity, offering promising strategies to improve food safety and reduce health risks associated with mycotoxin exposure.\u003c/p\u003e","manuscriptTitle":"Lutein and Zeaxanthin Provide Protection Against Aflatoxin B1-Induced Liver and Kidney Toxicity: Insights from in Silico and in Vivo Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-25 20:24:29","doi":"10.21203/rs.3.rs-8340180/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-24T08:41:40+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-18T13:27:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-15T06:10:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-15T06:08:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycotoxin Research","date":"2025-12-11T21:43:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycotoxin-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myre","sideBox":"Learn more about [Mycotoxin Research](http://link.springer.com/journal/12549)","snPcode":"12550","submissionUrl":"https://submission.nature.com/new-submission/12550/3","title":"Mycotoxin Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6f236034-5213-4e56-9c49-ac9f55fdcaa2","owner":[],"postedDate":"December 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-16T23:23:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-25 20:24:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8340180","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8340180","identity":"rs-8340180","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}