Abstract Chromium (Cr) and arsenic (As) pose a threat to the exposed population, leading to various renal ailments. Although individual toxicity has been well investigated, little is known about their combined effects. In light of the mounting concern over the environmental impact of heavy metals, the current study investigated the potential benefits of the selected nutraceuticals, i.e., biochanin-A (BCA), coenzyme Q10 (CoQ10), and phloretin (PHL) in combined Cr + As intoxicated Swiss albino mice, providing a comprehensive understanding of the mechanism of action. During the two-week investigation, Cr (75 ppm) and As (100 ppm) were given orally to induce renal toxicity, and were simultaneously treated with BCA (50 mg/kg), CoQ10 (10 mg/kg), and PHL (50 mg/kg) intraperitoneally. The Cr + As-treated group showed an increase in kidney somatic index, metal burden, protein carbonylation, and malondialdehyde, along with a decrease in the activity of (superoxide dismutase, catalase, glutathione-S-transferase, reduced glutathione, and total thiol). Furthermore, DNA degradation, histology, and altered SIRT1/Nrf2/HO‑1/NQO1/SOD2/CYP1A1/KEAP1/CAS-8, and CAS-3 gene expressions corroborated the above findings. Alternatively, co-treatment with the selected antioxidants reversed the above mentioned parameters, highlighting the protective effects of these compounds against Cr + As-induced oxidative damage. Nrf2, a key player in this process, is responsible for the activation of the antioxidant response element and subsequent expression of antioxidant enzymes. We further investigated the possible interactions of BCA, CoQ10, and PHL with the antioxidant enzymes/proteins, SIRT1/Nrf2/KEAP1/HO-1/NQO1, using in silico studies. Our study offers new avenues for the future of chronic kidney disease treatment associated with Cr + As-induced exposure, providing a deeper understanding of the role of Nrf2 in this context. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-025-13969-2. Keywords: Chromium, Arsenic, Reactive oxygen species, Biochanin-A, Coenzyme Q10, Phloretin Subject terms: Mechanisms of disease, Toxicology, Experimental models of disease Introduction Compounds containing chromium (Cr) and arsenic (As) are pervasive environmental toxins that present serious health risks worldwide, particularly in developing nations. Prior findings suggest that individuals exposed to the specific hexavalent chromate complexes and arsenic compounds through drinking water and industrial settings have experienced adverse health effects^[36]1. Numerous sectors, including the wine industry, metallurgy, wood preservatives manufacturing and application, and semiconductor manufacturing industries, expose workers to an amalgam of both toxicants. The general population is susceptible to exposure to metals from tainted food and water simultaneously. There is ample proof that chromium and arsenic seep through wood treated with copper, chromium, and arsenic contaminate the soil and groundwater^[37]2. Similar findings were made by Maduabuhci et al.^[38]3 on excessive levels of chromate and arsenic in Nigerian beverages. Prior studies have documented a multi metal co-contamination including chromium and arsenic in Indian groundwater with attendant grave health implications for the local residents^[39]4,[40]5. According to reports, both are present together in tainted groundwater and surface waterways^[41]6. Regarding their co-exposure in water, there are numerous papers available^[42]7,[43]8. Surface water was found to contain 21,800 µg/L of chromium and 86,100 µg/L of arsenic in a meta-analysis reported in a previous study^[44]9. According to a different investigation, surface water bodies had 1798 µg/L of chromium and 290.55 µg/L of arsenic^[45]10. Despite the fact that they coexist, there are less reports on their combined toxicity. These findings emphasize the pervasiveness of chromium and arsenic co-contamination in India as well as globally across both industrialized areas and rural regions. The body requires the essential micronutrient Cr [III] and, if present in deficient levels, may cause problems with lipid and sugar metabolism and increase the risk of cardiovascular diseases^[46]11. Due to its inability to enter cells and insufficient intracellular aggregation, it is considered a dietary supplement^[47]11,[48]12. On the other hand, the sulphur anion transport pathway allows Cr [VI] to swiftly traverse the plasma membrane^[49]13. Since the kidney is the main organ where chromium accumulates, it is more vulnerable to its damaging effects than other tissues^[50]14. Acute renal failure caused by the administration of potassium dichromate (K[2]Cr[2]O[7]), a Cr [VI] compound, is harmful to the kidney and appears to be reversible^[51]15. The proximal convoluted tubules (PCT) region and brush border membrane lining the epithelial cells are particularly impacted by K[2]Cr[2]O[7] exposure^[52]16. Arsenic, which can be identified in organic as well as inorganic forms (such as trivalent arsenite and pentavalent arsenate), constitutes one of the most significant environmental toxins. People’s health is impacted by inorganic arsenic, which is more poisonous than organic arsenic. Arsenic poisoning can harm the kidneys if ingested, whether through food, water, or medicine. As reported, 60% of the daily intake of arsenic, including inorganic and methylated, is eliminated in the urine, making the kidney a target organ^[53]17. It has been discovered that long-term exposure to arsenic alters the kidney, resulting in interstitial nephritis, glomerular hypertrophy, and tubular cell vacuolation^[54]18. The first anatomical region subjected to filtered toxicants, the PCT with epithelial cells with resorptive capacity, is the most vulnerable region of the kidney^[55]19. Chromium and arsenic enhances the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H[2]O[2]) and superoxide (O^− 2), according to studies conducted at the cellular and molecular levels^[56]20–[57]22. Arsenic has also been linked to increased protein oxidation, DNA and enzyme damage, and the induction of apoptosis^[58]23,[59]24. A few studies in the recent past have addressed regarding the alteration of the antioxidant machinery resulting in the increased oxidative stress in various tissues of the experimental model exposed to various heavy metals including cadmium, chromium and arsenic exposure^[60]25–[61]28. A study in past showed that the primary mechanism of arsenic pathogenesis is linked to decreased antioxidant capacity, which results in dysfunctional cellular macromolecules^[62]29. A member of the sirtuin family, silent information regulator 1 (SIRT1) is a histone deacetylase that primarily binds to nicotinamide adenine dinucleotide (NAD+), which is located in the nucleus. Because SIRT1 modulates most of the physiological events in the body, it interacts with protein substrates in different signalling pathways^[63]30,[64]31. The basic cellular metabolism is controlled by a protein known as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) which is an essential transcription factor and reduces oxidative stress by regulating the expression pattern of antioxidants and phase II detoxifying enzymes^[65]32. Additional functions of Nrf2 include scavenging ROS and lowering lipid and protein oxidation^[66]33. Kelch-like Ech-associated Protein-1 (KEAP1) keeps Nrf2 tethered in the cytoplasm, where it is degraded in an unstressed state by the ubiquitin–proteasome system. In the presence of oxidative stress, SIRT1 promotes the deacetylation and stabilization of Nrf2 and facilitates its nuclear translocation and transcriptional induction of antioxidant response genes. In parallel, oxidative modifications of KEAP1 cysteine residues cause the disruption of Nrf2 from KEAP1 so that Nrf2 can build up and stimulate cytoprotective pathways^[67]34,[68]35. After that, Nrf2 enters the nucleus, attaches itself to antioxidant response elements (AREs) found in the downstream target genes’ 5′-flanking promoter/enhancer region, and starts the transcription of those genes. The xenobiotic metabolising enzymes (XMEs) that are associated with cellular defence against toxicant-induced oxidative stress, such as NAD(P)H quinone oxidoreductase 1 (NQO1), Heme oxygenase 1 (HO-1), Superoxide dismutase (SOD), and Cytochrome P450, family 1, subfamily A, polypeptide (CYP1A), are expressed differently when Nrf2 is expressed^[69]36. According to Siegel et al. (2004), NQO1 is a Phase II XME that guards against both endogenous and exogenous quinones, quinone-imines, etc^[70]37. According to Ross and Siegel (2021) it is both triggered by the aromatic hydrocarbon receptor (AhR) directed response pathway and induced as a component of the Nrf2-mediated cellular stress response^[71]38. Another significant Nrf2 target gene is HO-1 provides defence against oxidative damage and has outstanding antioxidant qualities^[72]39. O[2]^− are converted to H[2]O[2] and O[2] by mitochondrial or manganese SOD (MnSOD). O[2]^− produce hydroxyl radicals (OH^−) through Fenton reaction and further glutathione peroxidase (GPx) and CAT converts H[2]O[2] into water and O[2], protecting the cell from oxidative damage. Phase I XME CYP1A1 changes both exogenous and endogenous substances into more polar, water-soluble variants^[73]25. Furthermore, high ROS levels trigger the apoptotic cascade by cleaving caspase 8 (CAS-8) and subsequently activating caspase 3 (CAS-3), which in turn degrades nucleic acids^[74]40. The utilization of dietary nutraceuticals with salutary value, free radical scavenging action, and antioxidant qualities against chromium and arsenic-induced nephrotoxicity has grown in popularity. Biochanin-A (BCA), coenzyme Q10 (CoQ10), and phloretin (PHL) are a few bioactive nutraceuticals that have antioxidant characteristics that scavenge free radicals and preserve cellular equilibrium^[75]41–[76]43. BCA also exerts a neuroprotective effect against L-glutamate-induced cytotoxicity, which plays a crucial role in neuronal cell death in various neurodegenerative diseases^[77]44,[78]45 while PHL, a β-dicarbonyl enols scavenges electrophile metal ions & unsaturated aldehyde through their nucleophile enolate form and becomes a rational candidate for treatment of oxidative stress linked disorders^[79]45,[80]46. Another antioxidant enzyme CoQ10 is lipid soluble and contributes to Electron Transport Chain (ETC) responsible for aerobic respiration^[81]47. Past findings have suggested that BCA and the combination of multiple doses of epigallocatechin gallate (EGCG) and CoQ10 mitigated the cisplatin-mediated pathogenesis by enhancing renal oxidative/nitrosative status, inflammation, and apoptosis^[82]48,[83]49. PHL also has nephroprotective effects and is helpful in the treatment of hyperuricemia-related renal disorders since it simultaneously inhibits nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) and uric acid reabsorption and attenuates uric acid-induced renal impairment^[84]50. We found the remedial potential associated with these compounds individually based on the previous findings. Every compound is unique in its bioavailability as per the structure and molecular weight, so we thought of giving them in the combination to see the synergic effects of these compounds in counteracting the nephrotoxicity associated with Cr + As as they have all shown the free radicals scavenging potential. Building on previous findings, the current research aimed to assess the protective roles of BCA, CoQ10, and PHL against oxidative stress, nephritic apoptosis, and molecular disruptions caused by simultaneous chromium and arsenic exposure in Swiss albino mice. The biochemical, histological, and molecular analysis not only aligns with the in-silico analyses but also serves as a strong validation of our predictions about the direct interaction of the BCA, CoQ10, and PHL with the transcription factor Nrf2 and its related XMEs. This alignment of different analyses provides a comprehensive view of the protective roles of these compounds, with potential implications for future research and clinical applications. It was assumed that simultaneous co-exposure with toxicants would enhance oxidative damage and inhibit Nrf2-dependent antioxidant responses. In contrast, treatment with natural compounds would counteract these effects by inducing the Nrf2/KEAP1 signaling pathway and reinforcing antioxidant defenses. The pictorial summary of the complete study is depicted in Fig. [85]1. Fig. 1. [86]Fig. 1 [87]Open in a new tab Overview pictorial summary of the study. Materials and methods Chemicals and reagents The chemicals such as sodium arsenite (NaAsO[2]), K[2]Cr[2]O[7], BCA, CoQ10, and PHL were obtained from Sigma, USA and additional chemicals essential for the experimental examination were procured from the different standard companies. Ethical statement All experimental protocols and methods were approved by the Institutional Animal Ethics Committee (IAEC) of ICMR-NIOH wide approval no. IAEC/NIOH/2018-19/21/02/M in compliance with the ARRIVE guidelines. Animals monitoring Swiss albino male mice (Mus musculus), weighing 25–27 g, were acquired from the Zydus Pharmaceutical Research Centre, Ahmedabad, Gujarat. The animals were kept in polypropylene cages for two weeks to help them get used to the laboratory environment. The mice were housed in a steady climate of 23 ± 2 °C with relative humidity of 55–60% and a 12-hour cycle of light and darkness. They were given unlimited access to purified water and the usual dry feeding pellets. Experimental design We chose to conduct our study for two weeks in order to examine the combined nephrotoxic effects of chromium and arsenic as well as the therapeutic effects of the specified nutraceuticals. The chosen toxicants were given through drinking water while the chosen antioxidants (BCA, CoQ10, and PHL) were given intraperitoneally during the entire research. The toxicants and natural compounds doses were chosen from previously published studies with a few minor modifications to the administration route^[88]51–[89]56, and with a meticulous pilot study conducted in our lab^[90]41,[91]57. In experimental toxicology, such high dosages are used to monitor dose-response relationships and possible side effects at harmful exposure levels. By using this method, we may examine the effectiveness of the chosen nutraceuticals in reducing the toxicity(s) linked to these heavy metal exposures and produce quantifiable toxic effects in the Swiss albino mouse model. The mouse model is essential to this practical application of our research, as high dosages are employed in experimental toxicology to evaluate the possible health effects. This emphasises the necessity of our research in creating efficient mitigation techniques. Our study’s validity and credibility are guaranteed by its reliance on well-established research. Figure [92]2 shows the experimental design of the study. Fig. 2. [93]Fig. 2 [94]Open in a new tab The flowchart of the experimental design. The mice were euthanized through cervical dislocation after being anesthetized by CO[2] inhalation after the treatment period was over. The kidney tissues were then separated, weighed, and homogenized in a solution of 100mM phosphate buffer and 0.1% TritonX-100 for all of the biochemical investigations. The kidney somatic index was calculated using: graphic file with name d33e579.gif Estimation of chromium and arsenic accumulation in kidney tissues To evaluate the quantity of chromium and arsenic in the kidney tissues, 150–200 mg of tissue were taken and acid digested in the TFM vessel through the Start ‘D’ Microwave Digester system, Milestone, Italy. Afterward, the digested samples were introduced into Atomic Absorption Spectrophotometer (Model AA-800, PerkinElmer, USA) to measure the total chromium and arsenic content in µg /g tissue weight. Estimation of biochemical parameters The activity of superoxide dismutase (SOD)^[95]58, catalase (CAT)^[96]59, glutathione-S-transferase (GST)^[97]60, reduced glutathione (GSH)^[98]61, total thiol (TT)^[99]62, protein carbonyl content (PCC)^[100]63, and lipid peroxidation (LPO)^[101]64 were assessed as per the described protocols respectively through Synergy H1MD, BioTek, USA Multimode analyser. DNA separation and gel electrophoresis of kidney tissues The kidney tissue’s chromosomal DNA (gDNA) was removed using the phenol/chloroform process. The pellets were then precipitated using ethanol and sodium acetate, and later, DNA quantitation was quantitated at 260/280 nm for purity. The isolated DNA was then loaded onto a 1% agarose gel and electrophoresed at 100 V for 30 min to check the integrity. Histopathology of kidney tissues With expert precision in context with the histological evidence, kidney tissues were first fixed in 10% neutral buffered formalin for 10 days and later transferred to 70% ethanol for further processing. Subsequently, kidney tissues were dehydrated via a serial ethanol gradient and embedded in paraffin wax blocks for further staining. Tissue section (5-mm-thick) were prepared, dewaxed in xylene, rehydrated via decreasing concentrations of ethanol, and washed with PBS. Tissue sections were then stained with hematoxylin and eosin (H&E) to detect any morphological defects by using light microscopy as described earlier^[102]65. Quantitative real time PCR (qRT-PCR) The RNA from kidney tissues was extracted using the Nucleospin RNA Plus kit. RNA quantification was done by measuring the absorbance at the 260/280 ratio and further it was reverse-transcribed into cDNA and later RT-qPCR was done to amplify and analyse cDNA using specific gene primers and SYBR green (detection dye). The gene expression values were calculated using the 2^−ΔΔCT method. Table [103]1 provides a list of the primer sequences that were employed to examine various genes. Table 1. List of qRT- PCR primers. Gene Forward Reverse SIRT1 ACCAAATCGTTACATATTCC CAAGGGTTCTTCTAAACTTG Nrf2 CCAGAAGCCACACTGACAGA GGAGAGGATGCTGCTGAAAG HO-1 GCCGAGAATGCTGAGTTCATG TGGTACAAGGAAGCCATCACC NQO1 TTCTGTGGCTTCCAGGTCTT AGGCTGCTTGGAGCAAAATA SOD2 TAACGCGCAGATCATGCAGCTG AGGCTGAAGAGCGACCTGAGTT CYP1A1 CTATCTCTTAAACCCCACCCCAA CTAAGTATGGTGGAGGAAAGGGTG KEAP1 GGCAGGACCAGTTGAACAGT GGGTCACCTCACTCCAGGTA CAS-8 TGCTTCATCTGCTGTATCCTATCC TGTTCCTCCTGTCGTCTTTATTGC CAS-3 CACTGGAATGTCATCTCGCTCTG GTCCCACTGTCTGTCTCAATGC GAPDH CATCACTGCCACCCAGAAGACTG ATGCCAGTGAGCTTCCCGTTCAG [104]Open in a new tab Cheminformatics analysis and absorption, distribution, metabolism, and excretion and toxicity (ADMET) profiling The SMILES formats of the chosen biocompounds were downloaded from the PubChem database and the chemoinformatics analysis was performed on BCA, CoQ10, and PHL through SwissADME ([105]http://www.swissadme.ch/) and Way2Drug ([106]https://www.way2drug.com/passonline/predict.php) and ADMET profiling of the biocompounds was done through pkcsm ([107]https://biosig.lab.uq.edu.au/pkcsm/prediction) online tools accordingly. Molecular docking, protein-protein interactions (PPI) and gene ontology (GO) The targeted amino acid sequences such as, SIRT1, Nrf2, KEAP1, HO‑1, and NQO1 were retrieved from the UniProt database ([108]http://www.uniprot.org/), while the ligands (BCA, CoQ10, and PHL) were downloaded from the PubChem database. The SMILES formats of the chosen biocompounds were converted to 3D structures for the analyses. Following that, the molecular docking of selected ligand-compound against modeled 3D structure of targeted proteins was performed using the Pymol Molecular Graphics System, Version 3.0, Schrödinger. The docking score and binding affinity of the ligands have been determined using system suitability options using default settings. PPI data were retrieved using the STRING database (version 12) ([109]https://string-db.org/). The targeted genes IDs were subjected to the ShinyGO v0.80 database ([110]https://bioinformatics.sdstate.edu/go/) to obtain GO annotation for biological processes. As per the previously described analysis, the selected gene set was entered in Enrich-KG ([111]https://maayanlab.cloud/enrichr-kg), and were used for gene set enrichment analysis to discover the crucial molecular signaling pathways^[112]66. Further, the enrichment results were visualized in a bar chart highlighting the top pathways based on –log10 adjusted p-values^[113]67. Statistical evaluation All the experiments were conducted at least three times to ensure the reproducibility of the results. The data obtained is stated as mean ± SEM. Results were analysed using a one-way analysis of variance test (one-way ANOVA) to compare differences against multiple groups, followed by a Newman-Keuls post hoc test to identify specific group differences. The minimal statistical significance was accepted at p < 0.05 with a 95% confidence interval. The statistical analyses were computed using the widely accepted and trusted GraphPad Prism (USA) software, known for its robust statistical capabilities and user-friendly interface, ensuring the validity of our results. The details are provided in each figure’s legend (wherever applicable). Figures were created using the industry-standard tools Adobe Illustrator CS5.1 (Adobe Systems Inc.) and PowerPoint 2016 (Microsoft), assuring the quality of the visual representations. Results Assessment of KSI According to our study, individual and combined intoxication of chromium and arsenic dramatically raised the KSI as compared to group 1, the control. However, it was found that all of the natural compounds (BCA, CoQ10, and PHL) that were chosen could restore kidney weight and body weight, which led to a drop in the somatic index. (Table [114]2). Table 2. Kidney somatic index (KSI) amid all the groups (Group 1: control; group 2: cr; group 3: as; group 4: Cr + as; group 5: Cr + As + BCA; group 6: Cr + As + CoQ10; group 7: Cr + As + PHL; group 8: Cr + As + BCA + CoQ10 + PHL). Groups Body Weight (g) ± SEM Kidney Weight (g) ± SEM Kidney Somatic Index (%) Control 33.30 ± 0.65 0.45 ± 0.035 1.35 ± 0.13 Cr 28.98 ± 1.36 0.46 ± 0.15 1.59 ± 0.26 As 28.70 ± 0.89 0.46 ± 0.06 1.60 ± 0.31 Cr + As 26.52 ± 0.41 0.47 ± 0.017 1.77 ± 0.07 Cr + As + BCA 33.30 ± 0.56 0.33 ± 0.050 1.00 ± 0.29 Cr + As + CoQ10 32.56 ± 0.67 0.35 ± 0.082 1.07 ± 0.17 Cr + As + PHL 32.54 ± 0.80 0.37 ± 0.137 1.13 ± 0.42 Cr + As + BCA + CoQ10 + PHL 36.74 ± 0.27 0.34 ± 0.028 0.93 ± 0.07 [115]Open in a new tab In parallel, there was a slight increase in the kidney weight due to Cr + As induced organomegaly in the tissue as compared to the sham control. Though, co-treatment with the different natural compounds restored the kidney weight as compared to the Cr + As toxicant group, although it was not found to be significant (Fig. [116]3). Fig. 3. [117]Fig. 3 [118]Open in a new tab Kidney weight in experimental groups (Group1: control; Group 2: Cr; Group 3: As; Group 4: Cr + As; Group 5: Cr + As + BCA; Group 6: Cr + As + CoQ10; Group 7: Cr + As + PHL; Group 8: Cr + As + BCA + CoQ10 + PHL). Values represented as the mean ± SE. Quantification of chromium and arsenic in kidney tissues The results indicated that the concentrations of chromium and arsenic were significantly higher in the combined toxicant group 2 compared to the untreated control group (1) Nonetheless, with the administration of the natural compounds in combination (group 3), the accumulation of chromium and arsenic was found to decrease significantly in contrast with the combined toxicant group (2) Also, our analysis revealed that concentration of chromium and arsenic was found to be significantly higher in the combined nutraceuticals group 3 as compared to the untreated control. It appears that the selected bioactive compounds in combination could mitigate the renal toxicity by lowering the concentrations of chromium and arsenic in the kidney of Swiss albino mice (Fig. [119]4). Fig. 4. [120]Fig. 4 [121]Open in a new tab Chromium (Cr) and arsenic (As) concentration evaluation amid all groups (Group 1: control; Group 2: Cr/As; Group 3: Cr + As + BCA + CoQ10 + PHL). Expressed values as the mean ± SEM; wt: weight. Values represented as the mean ± SE; * indicates significant difference from Cr + As co-exposed group, ^# indicates significant difference from Cr + As + BCA + CoQ10 + PHL. The significant difference is set at p < 0.05. Effects of Cr + As and BCA, CoQ10, and PHL treatment on oxidative stress indices Chromium and arsenic disrupt redox balance, which leads to biochemical disturbance and subsequently results in cell death. Our research demonstrates how Cr + As intoxication significantly increases ROS, and how oxidative stress is subsequently reduced by the administration of the chosen natural compounds (BCA, CoQ10, and PHL). Overall, it was seen that the individual as well as the combined chromium and arsenic intoxication decreased the activity of SOD, CAT, GST, GSH, and TT along with the increase in the activity of PCC and LPO. The three antioxidants (BCA, CoQ10, and PHL) were found to be effective at restoring the activity of these antioxidant markers, demonstrating their therapeutic benefits. The activity of SOD, CAT, GST, GSH, and TT was found to decrease in the chromium and arsenic intoxicated groups 2, 3, and 4 in comparison with the sham control respectively. Alternatively, the levels were found to increase with the administration of combined bioactive compounds (BCA, CoQ10, and PHL) in group 8 as compared to the (Cr + As) group 4. Moreover, the activity levels of SOD, CAT, GST, GSH, and TT were found to be significant in group 8 as compared to Cr + As group 4 (Figs. [122]5, [123]6, [124]7 and [125]8, and [126]9) respectively. Fig. 5. [127]Fig. 5 [128]Open in a new tab SOD estimation in experimental groups (Group1: control; Group 2: Cr; Group 3: As; Group 4: Cr + As; Group 5: Cr + As + BCA; Group 6: Cr + As + CoQ10; Group 7: Cr + As + PHL; Group 8: Cr + As + BCA + CoQ10 + PHL). Values represented as the mean ± SE; * indicates significant difference from Cr + As co-exposed group. The significant difference is set at p < 0.05. Fig. 6. [129]Fig. 6 [130]Open in a new tab CAT estimation in experimental groups (Group1: control; Group 2: Cr; Group 3: As; Group 4: Cr + As; Group 5: Cr + As + BCA; Group 6: Cr + As + CoQ10; Group 7: Cr + As + PHL; Group 8: Cr + As + BCA + CoQ10 + PHL). Values represented as the mean ± SE. * indicates significant difference from Cr + As co-exposed group. The significant difference is set at p < 0.05. Fig. 7. [131]Fig. 7 [132]Open in a new tab GST estimation in experimental groups (Group1: control; Group 2: Cr; Group 3: As; Group 4: Cr + As; Group 5: Cr + As + BCA; Group 6: Cr + As + CoQ10; Group 7: Cr + As + PHL; Group 8: Cr + As + BCA + CoQ10 + PHL). Values represented as the mean ± SE; * indicates significant difference from Cr + As co-exposed group. The significant difference is set at p < 0.05. Fig. 8. [133]Fig. 8 [134]Open in a new tab GSH estimation in experimental groups (Group1: control; Group 2: Cr; Group 3: As; Group 4: Cr + As; Group 5: Cr + As + BCA; Group 6: Cr + As + CoQ10; Group 7: Cr + As + PHL; Group 8: Cr + As + BCA + CoQ10 + PHL). Values represented as the mean ± SE; * indicates significant difference from Cr + As co-exposed group. The significant difference is set at p < 0.05. Fig. 9. [135]Fig. 9 [136]Open in a new tab TT estimation in experimental groups (Group1: control; Group 2: Cr; Group 3: As; Group 4: Cr + As; Group 5: Cr + As + BCA; Group 6: Cr + As + CoQ10; Group 7: Cr + As + PHL; Group 8: Cr + As + BCA + CoQ10 + PHL). Values represented as the mean ± SE; * indicates significant difference from Cr + As co-exposed group. The significant difference is set at p < 0.05. An increase in PCC levels was observed in the individual and combined chromium and arsenic intoxicated groups as compared to the sham control, but was found significant in the co-exposed (Cr + As) group 4. However, treatment with the different antioxidants reversed the effects of the toxicants, and restored the PCC levels significantly in the BCA, CoQ10, and PHL treated groups respectively along with the co-treatment group 8 compared to the combined toxicant (Cr + As) group 4. Also, there was a significant reduction in the PCC level in the combined antioxidants group 8 in contrast to the arsenic treated group 3 (Fig. [137]10). Fig. 10. [138]Fig. 10 [139]Open in a new tab PCC estimation in experimental groups (Group1: control; Group 2: Cr; Group 3: As; Group 4: Cr + As; Group 5: Cr + As + BCA; Group 6: Cr + As + CoQ10; Group 7: Cr + As + PHL; Group 8: Cr + As + BCA + CoQ10 + PHL). Values represented as the mean ± SE; * indicates significant difference from Cr + As co-exposed group, ^@ indicates significant difference from As exposed group. The significant difference is set at p < 0.05. An increase in LPO levels was observed in the individual and combined chromium and arsenic intoxicated groups, as compared to the sham control, but was found non-significant. However, treatment with the different antioxidants reversed the effects of the toxicants, and restored the LPO levels significantly in the co-treatment group 8 compared to the combined toxicant (Cr + As) group 4 (Fig. [140]11). Fig. 11. [141]Fig. 11 [142]Open in a new tab LPO estimation in experimental groups (Group1: control; Group 2: Cr; Group 3: As; Group 4: Cr + As; Group 5: Cr + As + BCA; Group 6: Cr + As + CoQ10; Group 7: Cr + As + PHL; Group 8: Cr + As + BCA + CoQ10 + PHL). Values represented as the mean ± SE; * indicates significant difference from Cr + As co-exposed group. The significant difference is set at p < 0.05. Isolation and quantitation of DNA The differences in DNA yield between the treatment groups are apparent in the gel picture. The bands show that, as compared to the other treatment groups, the combined intoxicated (Cr + As) group 2 had lower DNA yield and showed degradation. On the other hand, it was found that all the other treated groups exhibited an increase in DNA yield (Fig. [143]12). Fig. 12. [144]Fig. 12 [145]Open in a new tab DNA gel electrophoresis (in experimental groups (Group1: control; Group 2: Cr + As; Group 3: Cr + As + BCA; G4: Cr + As + CoQ10; G5: Cr + As + PHL; G6: Cr + As + BCA + CoQ10 + PHL). Histological evaluation Figure [146]13 depicts the H&E stained kidney segments. The kidney of the control mice did not exhibit any notable histological alteration (Fig. [147]13A). In Cr + As treated group, the kidney revealed minimal mononuclear cell infiltration (Fig. [148]13B). Mice treated with Cr + As + BCA, Cr + As + CoQ10, and Cr + As + PHL showed almost normal structure and occasional mononuclear cell infiltration (Fig. [149]13C and D, and [150]13E) respectively. In (Fig. [151]13F), Cr + As + BCA + CoQ10 + PHL mice group indicated nearly normal appearance with improved morphology of the cells and fewer lesions. Fig. 13. [152]Fig. 13 [153]Open in a new tab Histopathology of kidney sections. Figure 13A-F represents photomicrograph of each experimental group. (Group1: control; Group 2: Cr + As; Group 3: Cr + As + BCA; Group 4: Cr + As + CoQ10; Group 5: Cr + As + PHL; Group 6: Cr + As + BCA + CoQ10 + PHL). Arrows indicate mononuclear cell infiltration. Original magnification-200X. Alterations in the gene expressions of SIRT1, Nrf2, HO‑1, NQO1, SOD2, CYP1A1, KEAP1, CAS-8, and CAS-3 The renal toxicity brought on by the combination of (Cr + As) and the therapeutic effects of the chosen natural substances (BCA, CoQ10, and PHL) were analyzed by the alterations in the mRNA expression levels of genes (SIRT1, Nrf2, HO‑1, NQO1, SOD2, CYP1A1, KEAP1, CAS-8, and CAS-3) associated with oxidative stress and allied pathways. The combined (Cr + As) group 2 exposed mice had lower levels of SIRT1, Nrf2, HO-1, NQO1, and SOD2 as compared to the untreated control. Consecutively, it was found that the expressions of the aforementioned genes were raised following 2 weeks of therapy with natural bioactive substances as compared to the co-exposed (Cr + As) group 2. However, a significant upregulation in the activity of SIRT1, Nrf2, HO-1, NQO1, and SOD2 was found in the combined antioxidant regimen group 6 in comparison with the (Cr + As) group 2 (Fig. [154]14a, b, c and d, and [155]14e) respectively. Also, SIRT1 and SOD2 activity was found to be significantly higher in the BCA treated group 3 as compared to the (Cr + As) group 2 respectively (Fig. [156]14a and e) respectively. While going through inter-treatment groups evaluation, the NQO1 expression was found to be significantly upregulated in the combined antioxidants group 6 compared to the Cr + As + BCA treated group 3 (Fig. [157]14d). Likewise, the SOD2 expression was found to be significantly upregulated in the combined antioxidants group 6 compared to the untreated control, Cr + As + BCA treated group 3, Cr + As + CoQ10 treated group 4, Cr + As + PHL treated group 5 (Fig. [158]14e). Fig. 14. [159]Fig. 14 [160]Fig. 14 [161]Open in a new tab Gene expression analyses in the kidney tissue of the experimental mice (a) SIRT1, (b) Nrf2, (c) HO-1, (d) NQO1, (e) SOD2, (f) CYP1A1, (g) KEAP1, (h) CAS-8, and (i) CAS-3. (Group1: control; Group 2: Cr + As; Group 3: Cr + As + BCA; Group 4: Cr + As + CoQ10; Group 5: Cr + As + PHL; Group 6: Cr + As + BCA + CoQ10 + PHL). Values represented as the mean ± SE; * indicates significant difference from Cr + As co-exposed group, ^# indicates significant difference from Cr + As + BCA + CoQ10 + PHL, ^$ indicates significant difference from Cr + As + BCA. The significant difference is set at p < 0.05. Alternatively, the mRNA expressions of CYP1A1, KEAP1, CAS-8, and CAS-3 were found to be upregulated in co-exposed (Cr + As) group 2 in comparison with the sham control. Nonetheless, a significant downregulation was observed in the activity of CYP1A1, KEAP1, CAS-8, and CAS-3 in the combined antioxidant regimen group 6 in comparison with the (Cr + As) group 2 (Fig. [162]14f, g and h, and [163]14i) respectively. In addition, CAS-8 activity was found to be significantly downregulated in the BCA treated group 3 and CoQ10 treated group 4 as compared to the (Cr + As) group 2 respectively (Fig. [164]14h). Also, CAS-3 activity was found to be significantly downregulated in the Cr + As + BCA treated group 3, Cr + As + CoQ10 treated group 4, Cr + As + PHL treated group 5 compared to the (Cr + As) group 2 respectively (Fig. [165]14i). While going through inter-treatment groups evaluation, the KEAP1 expression was found to be significantly downregulated in the combined antioxidants group 6 compared to the Cr + As + BCA treated group 3 (Fig. [166]14g). Likewise, the CAS-8 expression was found to be significantly downregulated in the combined antioxidants group 6 compared to the untreated control, Cr + As + CoQ10 treated group 4, and Cr + As + PHL treated group 5 (Fig. [167]14h). Similarly, the CAS-8 expression was found to be significantly downregulated in the Cr + As + BCA treated group 3 compared to the untreated control, Cr + As + CoQ10 treated group 4, and Cr + As + PHL treated group 5 (Fig. [168]14h). Also, the CAS-3 expression was found to be significantly downregulated in the combined antioxidants group 6 compared to the untreated control (Fig. [169]14i). Chemoinformatics analysis and ADMET profiling BCA, CoQ10, and PHL were chemoinformatically analyzed using a variety of computational techniques to evaluate their physicochemical characteristics, drug-likeness, biological activities, and molecular docking interactions. Lipinski’s Rule of Five was followed by BCA and PHL, suggesting good drug-likeness except CoQ10. While BCA and PHL showed superior aqueous solubility, CoQ10 had the largest molecular weight and logP, indicating significant lipophilicity (Table [170]3). Table 3. Chemoinformatics properties and biological activity of BCA, CoQ10, and PHL. S. No. Property BCA CoQ10 PHL Chemoinformatics properties of BCA, CoQ10, and PHL 1. Molecular Formula C[16]H[12]O[5] C[59]H[90]O[4] C[15]H[14]O[5] 2. Molecular Weight (g/mol) 284.26 863.34 274.27 3. LogP (Octanol/ Water Partition Coefficient) 2.44 17.85 1.93 4. Topological Polar Surface Area (TPSA, Å^2) 79.90 52.60 97.99 5. Number of Hydrogen Bond Donors 2 0 4 6. Number of Hydrogen Bond Acceptors 5 4 5 7. Number of Rotatable Bonds 2 31 4 8. Lipinski’s Rule of Five Complies (due to low molecular weight and LogP) Violates (due to high molecular weight and LogP) Complies (due to low molecular weight and LogP) 9. Bioavailability Score 0.55 NA 0.55 10. Drug-likeness Good (small molecule) Moderate (large molecule) Good (small molecule) 11. Predicted Antioxidant Activity High (polyphenolic structure) High (electron transport chain support) High (polyphenolic structure) Biological activity of BCA, CoQ10, and PHL (~*Pa values) 12. Free Radical Scavenging 0.52 0.69 0.38 13. Anti-oxidant 0.67 0.75 0.43 14. HMOX1 0.72 0.36 0.70 15. Cytoprotectant 0.50 0.40 0.60 16. Chemoprotective 0.64 0.41 0.30 [171]Open in a new tab NA- Not available; Pa- Probability of activity; *Closer the value of Pa to 1 greater the activity of compound. The ADMET profiling of the selected biocompounds revealed good pharmacokinetic and safety profiles (Table S1). When considering absorption, there was moderate water solubility by all the compounds, with PHL (− 3.077 log mol/L) more water soluble compared to BCA (− 3.735) and CoQ10 (− 3.255). Caco-2 permeability was greatest for CoQ10 (1.296 log Papp), reflecting effective transcellular transport, followed by BCA (0.897), while PHL exhibited relatively low permeability (− 0.325). Human intestinal absorption was great for BCA (93.03%) and CoQ10 (91.87%), whereas PHL exhibited moderate absorption (60.5%). BCA and PHL were forecast to be substrates for P-glycoprotein (P-gp), indicating possible efflux, and CoQ10 was not. Distribution-wise, BCA had more favorable blood-brain barrier (BBB) permeability (− 0.221 log BB) compared to CoQ10 (− 0.961) and PHL (− 0.927). Likewise, CNS permeability (log PS) was best for CoQ10 (− 1.176), indicative of possible CNS access, in comparison to BCA (− 2.115) and PHL (− 2.535). Metabolically, both BCA and CoQ10 were forecasted as CYP3A4 substrates, but only BCA as a possible CYP1A2 inhibitor. PHL neither acted as a substrate nor an inhibitor of the tested CYP enzymes. For excretion, CoQ10 had the highest calculated total clearance (1.345 log ml/min/kg), reflecting effective systemic removal, and BCA and PHL comparable and lower clearances (0.247 and 0.213, respectively). None of the compounds were recognized as renal OCT2 substrates, implying non-dependence on this transporter for renal elimination. Toxicity predictions showed good safety profiles for all compounds. The three compounds were not predicted to be mutagenic (AMES negative), hepatotoxic, or hERG I inhibitors by indicating low risk for genotoxicity, liver injury, or cardiotoxicity. Molecular docking of biocompounds and PPI Crucially, molecular docking studies revealed strong binding affinities for all drugs to well-known antioxidant-related proteins such as SIRT1, Nrf2, KEAP1, HO‑1, and NQO1. Binding energy (kcal/mol) and docking score of BCA, CoQ10, PHL against individual target proteins are presented in Tables [172]4 and [173]5. BCA at SIRT1 shows polar interaction with residues such as Arg 254, Ile 339, Asp 340, and Tyr 689 (Fig. [174]15a), whereas, CoQ10 shows these interaction with Arg 266 and Phe 265 (Fig. [175]15b), and PHL showed these interaction with Ile 339, Asp 506, and Val 404 (Fig. [176]15c). With Nrf2 the interaction of BCA were non-polar with residues such as Phe 149, Glu 156, Ser 333, Cys 506, Arg R, and Lys 510 (Fig. [177]15d). CoQ10 showed these interaction with Phe 116, Leu 123, Pro 141, Met 176, Trp 180, Leu 189, Leu 192, Phe 221, Phe 243, Phe 247, Phe 319, Leu 549, Leu 552, Val 556, Arg 561, and Asp 562 (Fig. [178]15e) and PHL interacted at Asp 16, Ser 342, Cys 506 and Lys 510 (Fig. [179]15f). BCA interact with KEAP1 having polar interaction ar Glu 528 (Fig. [180]15g) while CoQ10 had polar interaction with residues such as Arg483, Ser 508 and non-polar interaction with the residue Tyr 334, Arg 415 (Fig. [181]15h). PHL showed polar interaction with residues such as Arg 415, Arg 483, Ser 507 and Ser 555 of KEAP1 (Fig. [182]15i). HO-1 showed polar interaction with the residues like Glu 41, Ser 43 and non-polar interaction with the residues such as Phe 37, Val 42, and Phe 47 with BCA (Fig. [183]15j). CoQ10 at HO-1 shows non-polar interaction with the residues, such as Glu 29, Phe 33, Met 34, Val 50, Tyr 134, Thr 135, Arg 136, Ser 142, Leu 147, Phe 207, Asn 210, Leu 213, Thr 281, and Met 289 (Fig. [184]15k). PHL showed polar interaction with Pro 136, Asp 140 and shows non-polar interaction with the residues, such as Ala 28, Phe 37, and Ile 286 to HO-1 (Fig. [185]15l). Similarly, BCA at NQO1 shows polar interaction with the residues such as Trp 105, Phe 107, Tyr 156, His 162 and non-polar interaction with the residue Trp 162 (Fig. [186]15m). The receptor binding pocket of CoQ10 at NQO1 shows polar interaction with the residue His 162 (Fig. [187]15n), whereas, PHL interacted with NOQ1 at Phe 107 and His 162 (Fig. [188]15o). Table 4. Binding energy for the redox regulatory and other studied proteins. Target Binding energy (kcal/mol) BCA CoQ10 PHL SIRT1 -36.765 NS -30.12 Nrf2 -16.098 -26.972 -27.84 KEAP1 -24.442 NS -37.387 HO-1 -26.661 NS -43.138 NQO1 -32.547 -56.826 -36.988 [189]Open in a new tab NS- not suitable (was not suitable for docking). Table 5. Docking score for the redox regulatory and other studied proteins. Target Docking score BCA CoQ10 PHL SIRT1 -7.045 NS -8.496 Nrf2 -1.516 0.893 -2.919 KEAP1 -0.691 NS -5.119 HO-1 -4.271 NS -6.98 NQO1 -4.404 -1.019 -5.526 [190]Open in a new tab NS- not suitable (was not suitable for docking). Fig. 15. [191]Fig. 15 [192]Open in a new tab In silico binding of BCA, CoQ10, PHL (a) Docked conformation of BCA, CoQ10, PHL ligand at the binding site of SIRT1 receptor. (b) Docked conformation of BCA, CoQ10, PHL ligand at the binding site of Nrf2 receptor. (c) Docked conformation of BCA, CoQ10, PHL ligand at the binding site of KEAP1 receptor. (d) Docked conformation of BCA, CoQ10, PHL ligand at the binding site of HO-1 receptor. (e) Docked conformation of BCA, CoQ10, PHL ligand at the binding site of NQO1 receptor. BCA, CoQ10, PHL ligands are shown in stick style and coloring scheme is atom type. The interacting amino acids in the receptor are shown in line style, and rest of the amino acid residues in secondary structure representation. The PPI networks for the chosen antioxidant, detoxification, and apoptosis-related proteins were mapped from the STRING database (Fig. [193]16). Panel (a): SIRT1 showed interactions with FOXO1, TP53, PPARG, CLOCK, and ARNTL, pointing towards its function in oxidative stress resistance and metabolic regulation. Panel (b): NFE2L2 (Nrf2) showed a dense interaction with KEAP1, HMOX1, NQO1, PARK7, and several MAF transcription factors, which points out the conventional Nrf2-KEAP1-ARE antioxidant pathway. Panel (c): KEAP1’s interactions with NFE2L2, CUL3, RBX1, and IKBKB indicate crosstalk between redox signaling and the ubiquitin-proteasome system. Panel (d): HMOX1 formed tight correlations with FECH, BLVRB, KEAP1, and NQO1, which further support its function as a stress-inducible antioxidant enzyme. Panel (e): NQO1 was associated with KEAP1, CRYZ, GCLC, TP53, and HMOX1, indicating its detoxifying role in oxidative protection. Panel (f): SOD2 was associated with GPX7, FOXO3, PPARGC1A, CAT, and PARK7, charting its role in mitochondrial ROS homeostasis. Panel (g): CYP1A1 was associated with other detoxification enzymes such as UGT1A isoforms, CYP3A4, GSTM1, and COMT, indicating its key position in the metabolism of xenobiotics. Panel (h): CASP8 had strong associations with FADD, TNFRSF10B, CFLAR, RIPK1, and CASP3, indicating the extrinsic pathway of apoptosis. Panel (i): CASP3, being an effector caspase, was functionally associated with CASP9, APAF1, CYCS, BIRC proteins, and PARP1, emphasizing its regulatory position in intrinsic apoptosis. Fig. 16. [194]Fig. 16 [195]Open in a new tab PPI analyses of target genes. Interactions between monitored proteins (a) SIRT1, (b) Nrf2, (c) HO-1, (d) NQO1, (e) SOD2, (f) CYP1A1, (g) KEAP1, (h) CAS-8, and (i) CAS-3 estimated using STRING 12 database. GO and network clustering of target proteins Functional enrichment analysis of the major antioxidant and apoptosis-associated targets regulated by CoQ10, BCA, and PHL identified some considerably enriched GO biological processes (Fig. [196]17). The network of interactions (left) shows close functional clustering of terms related to oxidative stress response, ROS management, and apoptotic control. The highest-enriched GO terms, as indicated by the dot plot (right), are removal of superoxide radicals, negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway, cellular response to superoxide, and response to hydrogen peroxide. Dot size indicates the number of genes under each term (from 3 to 6), whereas intensity of color represents statistical significance as –log10 (FDR). Significantly, the processes with the greatest fold enrichment and most statistical significance were specifically implicated in redox balance, mitochondrial stress adaptation, and modulation of cell death. Fig. 17. [197]Fig. 17 [198]Open in a new tab GO analysis using ShinyGo identified enriched biological processes of target genes. The green dots represent the nodes for each GO biological process, while the lines represent the interaction between the nodes (minimum of 20% genes common between two connected) GO processes. KEGG pathway enrichment analysis revealed significant enrichment in pathways associated with oxidative defense, detoxification, and programmed cell death. The analysis detected significantly enriched pathways and phenotypic associations using various databases, such as WikiPathways, DisGeNET, KEGG, Reactome, GO Biological Processes, and MGI Mammalian Phenotype. Each colored bar corresponds to a distinct pathway or phenotype term, with categories colored according to the database source (Supplementary Fig. [199]S2). The findings corroborate the implication of the Nrf2/HO-1 pathway and related stress-response pathways in mediating the toxicological effects observed. Supplementary Figure [200]S3 presents the top enriched pathways from our KEGG analysis, with a focus on apoptosis, Nrf2 signaling, xenobiotic metabolism, and other critical biological processes These pathways, ranked based on their statistical significance (adjusted p-values < 0.05), and their crucial relevance to oxidative stress and renal injury mechanisms, underscore the importance of our research. They could pave the way for future research in understanding and mitigating the effects of toxic insults on biological functions, highlighting the significance and relevance of our findings. Discussion In the present scenario, natural antioxidants are in high demand due to their enormous healing properties and better chelation; they are substituting conventional chelating agents to counteract heavy metal-associated toxicity. The goal of the current study was to gain a better knowledge of the oxidative damage that chromium and arsenic cause to the renal tissue, as well as the function of the chosen antioxidants (BCA, CoQ10, and PHL) play in reducing (Cr + As)-induced changes in experimental mice. In our study, there was a rise in the kidney weight and the KSI in the combined toxicant (Cr + As) group 4. The modest increase in KSI seen in the (Cr + As) treated mice could be attributed to an excessive buildup of chromium and arsenic in the renal tissue. Also, it was found that the selected antioxidants could mitigate the accumulated toxicants in the kidney tissues resulting in decreased KSI. A previous study has reported the same trend of KSI and accumulation of chromium and arsenic in the kidney tissues of the experimental animals^[201]68,[202]69. One of the main mechanisms of chromium and arsenic-mediated toxicity is oxidative stress, which can be attributed to their combined inhibitory effects on the antioxidant defense system and associated signaling pathways. The results of this investigation showed that (Cr + As) exposure led to the reduction of both enzymatic (SOD, CAT, GST) and non-enzymatic (GSH, TT) antioxidant molecules as well as an increase in the PCC and LPO levels in the renal tissue. Though the specific mechanisms are unknown, mounting evidence shows that (Cr + As) directly and indirectly cause ROS^[203]22,[204]27,[205]41. These results were concomitant with the previous studies that demonstrated that chromium and arsenic exposure causes a misbalance in the redox status, thus, generating ROS^[206]70,[207]71. The current investigation demonstrated that SOD and CAT inhibition by the combined toxicants (Cr + As) was significantly reversed by the chosen antioxidants (BCA, CoQ10, and PHL) treatment. In terms of radical formation, it has been demonstrated that the reactive intermediates of Cr[VI], i.e., Cr(V) or Cr(IV), and dimethyl arsine, a derivative of dimethyl arsenic acid, may react with the H[2]O[2] produced as a result of O^− 2 dismutation. Further, this H[2]O[2] is then degraded by CAT to H[2]O and O[2]. Hence, enzymatic antioxidants are necessary for effective defense counter to impairment caused by ROS. Our results were concurrent with the past findings where exposure to chromium and arsenic led to decreased SOD and CAT activity. At the same time, treatment with N-Acetyl-L-Cysteine (NAC) and resveratrol reversed the effects of the toxicants and reduced oxidative stress^[208]17,[209]72. A recent study showed that CoQ10 may successfully shield kidney and liver tissues from oxidative hepatotoxicity and nephrotoxicity brought on by carbofuran^[210]73. These findings highlight how natural antioxidants can be used therapeutically to preserve enzymatic redox equilibrium in the presence of metal-induced oxidation. Specifically, GSTs affect the binding of GSH to a broad spectrum of electrophilic complexes, and they are a group of phase II detoxifying enzymes that protect macromolecules in cells against ROS. In our study, a decrease in GST levels was found, and it might be brought on by GSH being consumed while providing defense against Cr + As generated oxidative stress since GSH functions as a cofactor for GST and is essential for avoiding Cr + As toxicity and carcinogenicity. On the other hand, it was found that the supplementation with chosen antioxidants (BCA, CoQ10, and CoQ10) effectively restored the GST levels to normal and thwarted the caused oxidative stress. The combined antioxidants group 8 showed a substantial increase compared to the toxicant group 4. Previous findings also suggest the ameliorative role of Fagonia indica and BCA, PHL, and EGCG in chromium and arsenic-induced toxicity in the experimental mice, respectively^[211]57,[212]74. This demonstrates how nutraceutical antioxidants can be combined to improve cellular detoxification and combat oxidative stress brought on by metals. Exposure to chromium and arsenic not only increases the production of pro-oxidants but also substantially reduces non-enzymatic antioxidant defences, especially GSH and TT, which are essential for preserving cellular redox equilibrium. One of the main sulfur-containing non-proteins, GSH is crucial for detoxifying electrophiles, scavenging ROS^[213]75, regulating protein activities, and regenerating other antioxidants such ascorbic acid^[214]76. Following Cr + As intoxication, our study showed a significant decrease in GSH levels, suggesting oxidative stress and weakened cellular defence. GSH levels were considerably raised by BCA, CoQ10, and PHL therapy, demonstrating their ability to combat oxidative stress and lessen nephrotoxicity. Similarly, the Cr + As group also showed a substantial reduction in protein thiols, which contain reactive (-SH) groups essential for shielding cells from oxidative damage. Heavy metal-induced oxidation primarily targets these thiol groups, resulting in protein malfunction, cellular instability, and ultimately cell death. Antioxidant therapy successfully raised TT levels in our study, particularly in the combination group, indicating a return to redox balance. These results are in line with earlier research that demonstrated thiol depletion in animal models exposed to chromium^[215]77 and arsenic^[216]78 respectively. However, treatment with folic acid and arjunolic acid altered the metabolic activity of the thiols, preventing the induced oxidative stress and sustaining the redox balance. Also, the selected compounds in this study have previously showed significant increase in thiol level in different tissues of experimental model^[217]22,[218]41,[219]42. Overall, the findings support the protective effect of BCA, CoQ10, and PHL, especially when combined, in preserving intracellular thiol status and averting renal oxidative damage brought on by Cr + As. An established indicator of protein oxidation is protein carbonylation. Carbonyl derivatives are created when free radicals interact with the side chains of the lysine, arginine, and glutamic acid residues of proteins^[220]79. Additionally, the lysine-amino group can form a bond with both reducing sugars and their byproducts of oxidation, such as hydroxynonenal (HNE) and malondialdehyde (MDA), or with the dialdehyde products of LPO, such as HNE^[221]80. The rise in the level of PCC in kidney tissues exposed to (Cr + As) may be due to either an excess of ROS production or a buildup of ROS brought on by the ineffectiveness of antioxidants after 2 weeks of exposure to chromium and arsenic. In the mice exposed to Cr + As but receiving selected antioxidants (BCA, CoQ10, and PHL), our current investigation revealed a significant decrease in the level of protein oxidation. O^− 2, hydroxyl (OH), and hydroperoxyl (HO[2]) radicals can be effectively neutralized by the chosen nutraceuticals. High interaction rates between (BCA, CoQ10, and PHL) and lipid peroxyl radicals interrupt the radical chain, which stops further LPO. Antioxidant supplementation to mice receiving Cr + As lowers protein oxidation by counteracting the ROS produced by the toxicants. Prior studies have demonstrated that exposure to chromium or arsenic increases the PCC levels^[222]22,[223]41. At the same time, supplementation with Fagonia indica and BCA, PHL, and EGCG decreased the PCC activity in the renal tissues^[224]57,[225]74. Several metals, particularly chromium, and arsenic, have been shown to produce reactive species that cause LPO and sulfhydryl depletion^[226]81. Previous research on chromium-exposed employees and experimental models have demonstrated that chromium and arsenic can cause LPO^[227]72,[228]82. Acute Cr(VI) exposure in rats has been linked to increased hepatic (70%) and renal (15%) LPO^[229]83. Regarding the potential significance of reactive intermediates, it has been demonstrated that generating Cr(V) and Cr(IV) molecules accelerates LPO in kidney homogenates. Free radicals injure cells through the process known as LPO, which occurs when they remove an electron from the lipids in plasma membranes. It usually impacts polyunsaturated fatty acids (PUFAs) because they have several double bonds and methylene bridges (-CH[2]) with reactive hydrogen atoms within them^[230]84. Previous studies have showed that treatment with BCA, CoQ10, or PHL have effectively reduced the MDA levels in singlet or combined exposure to chromium, arsenic, or other metals in different experimental models^[231]22,[232]27,[233]28. Our results are consistent with the above findings as Cr + As toxicity increased the LPO levels while treatment with the chosen antioxidants reduced the LPO activity. The light microscopic images of the medullar part of the kidney in the control mice showed normal renal structure, while the combined toxicant group showed minimum mononuclear infiltration. At the same time, co-treatment with BCA, CoQ10, or PHL and BCA + CoQ10 + PHL reduced the detrimental effects of combined toxicants (Cr + As) on the renal structures. This could indicate improved nephron function or reduced damage to renal cells in the presence of BCA + CoQ10 + PHL, respectively. These findings are consistent with the theory that chromium and arsenic cause renal toxicity by affecting biochemical mechanisms mediated through oxidative stress. Our results also exhibit the protective effects of selected natural compounds in mitigating the toxicity caused by chromium and arsenic in the kidneys. The outcomes of the biochemical stress variables showed that combined intoxication of chromium and arsenic, produce significant oxidative damage. Therefore, the goal of the current study was to comprehend how the antioxidant defence system functions under such complicated circumstances. After the treatment period, the mRNA expression pattern of the Nrf2- KEAP1-ARE pathway components and associated antioxidants, as well as XMEs (SIRT1, Nrf2, HO‑1, NQO1, SOD2, CYP1A1, KEAP1) and apoptosis indicators, CAS-8 and CAS-3 were seen, which is novel in our opinion. It is now widely accepted that Nrf2 signaling is a critical transcriptional regulatory system involved in the metabolism and detoxification of many toxins^[234]85. Our findings showed that Cr + As downregulated the mRNA expressions of SIRT1, Nrf2, HO-1, NQO1, and SOD2 and upregulated the levels of CYP1A1, KEAP1, CAS-8, and CAS-3 in comparison with the sham control, but supplementation of BCA, CoQ10, and PHL in combination significantly improved nephritic Nrf2 and its related XMEs. The Nrf2 expression pattern significantly increased, suggesting that BCA, CoQ10, and PHL had synergistic or additive effects. It is in concurrence with the previous studies, where authors have demonstrated that ellagic acid can reduce neuronal damage caused by oxidative stress through the activation of Nrf2^[235]86. After 15 days, KEAP1 expression was raised in the co-exposed (Cr + As) group; however, nutraceuticals administration significantly downregulated the KEAP1 expressions. Accordingly, an auto-regulatory feedback mechanism between KEAP1 and Nrf2 that controls their cellular stability is suggested by the enhanced KEAP1 expression seen in this work^[236]87. Under normal cellular homeostasis, Nrf2 is tightly bound to KEAP1 and is constantly polyubiquitinated and degraded by the proteasome via culin-3 (Cul3)-dependent E3 ubiquitin ligase^[237]88. The role of KEAP1 in this process is crucial, as it acts as a sensor for oxidative and electrophilic stress, a key regulatory mechanism in the Nrf2-ARE pathway. When the cell is under stress, KEAP1 disengages from Nrf2, allowing Nrf2 to be released and transcribed. This transcription triggers the production of downstream AREs^[238]89. Nrf2 expression is not just a function but a vital adaptive mechanism shielding cells from oxidative damage^[239]90. The disruption of Nrf2 signaling due to chromium and arsenic exposure is a serious issue, as it undermines the crucial role of Nrf2 in cellular defense, a role that cannot be overstated. Chromium and arsenic exposure can lead to the production of antioxidant enzymes, which are insufficient to counter the level of damage and cause a severe imbalance in the oxidation balance, further exacerbating the issue. Therefore, continuous chromium and arsenic co-exposure will eventually deplete these enzymes, reducing their activity and consequently, the activity of Nrf2, as demonstrated in an earlier study^[240]91. Moreover, chromium and arsenic exposure may contribute to the degradation of Nrf2 protein at a rate faster than its synthesis, leading to a decrease in its concentration within the nucleus. This phenomenon could be attributed to the disruption of intracellular oxidation balance caused by chromium and arsenic exposure, resulting in downregulation of the Nrf2 signaling pathway. Additionally, it has been observed that exposure to cadmium (Cd) increases KEAP1 expression in the kidney of Channa punctatus^[241]92. According to this study, in all the natural compounds treatment groups, Nrf2 expression levels were higher than those of the combined (As + Cr) group 2. In the combined nutraceuticals treatment group 6 there was maximum Nrf2 activation and nuclear localisation, thus representing the additive effects of the selected compounds. While synergism was noted in the combined anti-oxidants group, additive effects on NQO1 induction were observed in all the natural compounds treated groups. Since NQO1 has O[2]^− reductase activity and can provide further protection against oxidative damage, an increase in NQO1 mRNA level in treated groups implies that it is activated through Nrf2 to resist oxidative stress^[242]38. Compared to individual natural compounds treatment groups, the mixture group had much greater induction, indicating additive effects. Increased HO-1 expression in response to curcumin against aristolochic acid nephropathy^[243]93 and BCA against high-fat diet nephropathy^[244]94 are two examples of evidence supporting this conclusion. Our result is in concordance with the previous findings in an experimental model where Ipomoea staphylina and sulforaphane attenuated the chromium and arsenic-induced nephrotoxicity by up-regulating the HO-1 and Nrf2 gene, respectively^[245]95,[246]96. The increased SOD2 expression suggested the Nrf2 mediated activation of antioxidant defence physiological reaction. A key indicator of oxidative stress, CYP1A1 is a phase I detoxifying enzyme. In the current investigation, all the natural compounds treatment groups showed a downregulation of CYP1A1 expression after 15 days. This suggests that exposure to BCA, CoQ10, and PHL has activated the enzymatic system. Additionally, Wise et al. (2022) observed that following hexavalent chromium exposure, there was an increase in the CYP1A1 activity and protein expression in the immortalized human lung cells^[247]97. Apoptosis is a process that eliminates unwanted, damaged, or contaminated cells in various biological functions, including cell growth, differentiation, and proliferation in complex organisms^[248]98. Physical and chemical stimuli, including as heavy metals, xenobiotic agents, and OS, have been shown to induce apoptosis^[249]99. Mammals’ apoptotic response is mostly regulated by CAS-8 and CAS-3, which activate subsequent caspase enzymes^[250]100. The mitochondrial adenine nucleotide translocator is influenced by chemicals, such as As[2]O[3], which opens the mitochondrial permeability transition pores. As a result, the mitochondrial membrane degrades and more cytochrome c is released. DNA breakage, genomic instability, and cell death are the ultimate outcomes of the enhanced stimulation of CAS 3 brought on by the released cytochrome c^[251]101. Our findings demonstrated that, when compared to the sham control, the intoxication of Cr and As increased the expression of CAS-8 and CAS-3. In contrast to (Cr + As) group 2, the mRNA expression levels of CAS-8 and CAS-3 were reversed upon treatment with the chosen antioxidants (PHL, BCA, and CoQ10). Our results are in line with previous studies that demonstrated rats treated with CoQ10 and naringin decreased the effect on CAS-3 levels in the hepatic and renal tissues^[252]24. The application of cheminformatics in natural compound-based drug research has been explored recently and is used to screen compounds for various diseases robustly^[253]102. Cheminformatics and molecular docking are invaluable tools in compound-gene interaction; hence, we have conducted these studies in silico. The chemoinformatics analysis provided critical insights into the potential therapeutic interactions of BCA, CoQ10, and PHL with key antioxidant and cytoprotective proteins: SIRT1, Nrf2, KEAP1, HO-1, and NQO1. All three compounds demonstrated favorable physicochemical, drug-likeness, and biological activities, including Lipinski’s Rule of Five, indicating good drug-likeness, with the exception of CoQ10. This is in concurrence with a previous study that identified BCA as a potential candidate against breast cancer during docking studies with the target genes, further bolstering our optimism about the potential of these compounds^[254]103. While BCA and PHL showed superior aqueous solubility, CoQ10 had the largest molecular weight and logP, indicating significant lipophilicity. ADMET predictions for BCA, CoQ10, and PHL are valuable in terms of understanding their pharmacokinetic properties and safety profiles, and thus supporting them as therapeutic agents. All three nutraceuticals had acceptable water solubility and good intestinal absorption, with BCA and CoQ10 having very high absorption rates (> 90%) but PHL having moderate absorption. Even though PHL has poor Caco-2 permeability, its overall pharmacokinetic profile is still valuable owing to good solubility and non-toxicity. Distribution analysis indicated the best BBB permeability for BCA. It implied the potential for CNS accessibility, while CoQ10 had the best CNS penetration (log PS = − 1.176), which can increase its utility as a neuroprotectant. The fact that PHL and BCA are P-gp substrates, which can influence their bioavailability due to potential efflux, is a significant consideration. At the same time, the absence of CoQ10 as a P-gp substrate is beneficial for long-term systemic exposure. Metabolic profiling revealed both BCA and CoQ10 as potential CYP3A4 substrates, suggesting they can be subject to hepatic metabolism. BCA’s added function as an inhibitor of CYP1A2 might indicate some drug-drug interactions, which require in vitro confirmation. With little CYP interaction, PHL should be pharmacokinetically stable and less subjected to metabolic variability. Regarding excretion, CoQ10 had the greatest total clearance, indicating fast elimination, which might require repeated administration or formulation approaches to maintain the action. The lack of OCT2-mediated renal excretion in all compounds indicates that hepatic clearance would be the main avenue. Notably, neither of the compounds were AMES toxic, hepatotoxic, or hERG I inhibitory, highlighting their positive safety profiles with minimal predicted risks of genotoxicity, liver damage, or cardiotoxicity. These results confirm the further exploration of these compounds, most notably PHL and BCA, as multi-target antioxidants with therapeutic promise while also indicating the necessity for experimental confirmation and formulation optimization, particularly for poorly permeable or quickly cleared compounds. The binding affinities of BCA, CoQ10, and PHL against five important antioxidant-related targets- SIRT1, Nrf2, KEAP1, HO-1, and NQO1- were assessed using molecular docking. Stronger anticipated binding affinity is indicated by greater negative docking scores. Results show that BCA also had good interaction (− 7.045 kcal/mol), while PHL had the strongest binding affinity with SIRT1 (− 8.496 kcal/mol), indicating a high potential for influencing SIRT1-mediated pathways. Since CoQ10 was a bigger molecule, and due to docking restrictions or an unfavourable fit, CoQ10 was not given any binding score. Similarly, PHL demonstrated the strongest (although still poor) interaction (− 2.919 kcal/mol) with Nrf2, while the other three compounds showed weak binding to Nrf2. This implies that upstream modulators like KEAP1 may be more important than direct Nrf2 modulation as the primary mechanism^[255]104. PHL, in particular, showed a substantial interaction with KEAP1 (− 5.119 kcal/mol), suggesting a promising potential for PHL to inhibit KEAP1 and enhance Nrf2 activation. This finding is particularly hopeful, as it aligns with a similar trend observed in a study on the activation of Nrf2 with vitamin E and curcumin through NFĸB/AKT/mTOR/KEAP1 signalling in rat heart^[256]105. On the other hand, CoQ10 and BCA showed little to no binding. BCA (− 4.271 kcal/mol) and PHL (− 6.980 kcal/mol) exhibited significant binding with HO-1, indicating an efficient interaction with this downstream antioxidant protein. When it comes to NQO1 binding, PHL leads the pack with the best docking score of − 5.526 kcal/mol, followed by BCA (− 4.404 kcal/mol) and CoQ10 (− 1.019 kcal/mol). These findings suggest a potential impact on antioxidant defence through this target, sparking intrigue about the implications of this research. With its consistently strong binding affinity across all targets, particularly SIRT1, KEAP1, HO-1, and NQO1, PHL stands out as the most promising biocompound for modifying the Nrf2-mediated antioxidant defence system. Despite CoQ10’s low docking success, possibly due to its bulky and lipophilic nature that limits its fit within binding sites, BCA shows moderate action, especially with SIRT1 and NQO1, sparking intrigue about its potential in future research. Importantly, our findings align with the recent study, which used in silico/docking methods to study the activation of Nrf2/KEAP1 by the selected natural compounds, providing a strong foundation for our research^[257]106. In future research, these findings could be further explored to develop drugs that target the Nrf2-mediated antioxidant defence system. The PPI networks show that the molecular targets targeted by BCA, CoQ10, and PHL are closely associated with redox homeostasis, detoxification, and cell death pathways, which are of greatest relevance in countering chromium and arsenic-induced toxicity. The Nrf2-axis (panels b–e) verifies the functional ordering of antioxidant protection with KEAP1, HMOX1, and NQO1. These results confirm cheminformatics predictions and propose that all three compounds can act by potentiating the Nrf2-KEAP1-ARE signaling pathway. This is in agreement with a similar study performed in rats, where authors have shown the protective mechanism of ShenKang through the KEAP1/Nrf2/HO-1 signaling pathway in diabetic kidney disease through network pharmacology^[258]107. The docking affinity of PHL and BCA with these proteins, which is observed, is quite consistent with their centrality in the networks. SIRT1’s interaction with FOXO1, TP53, and circadian genes (panel a) suggests further layers of regulation, such as transcriptional adjustment to oxidative and metabolic stress- particularly pertinent to CoQ10’s function in mitochondrial metabolism. The mitochondrial antioxidant enzyme SOD2 (panel f), operationally connected with GPX7, CAT, and ACO1/2, serves to reinforce this connection. Detoxification pathways (panel g) focused on CYP1A1 showed broad interaction with phase II conjugate enzymes (UGTs, GSTs), implicating that BCA and PHL might be involved in xenobiotic elimination and heavy metal protection through increased biotransformation. Finally, the apoptosis modules (panels h and i) including CASP8 and CASP3, illustrate the ability of the compounds to modulate programmed cell death upon cellular injury. Such modulation is imperative in evading chronic inflammation and tissue damage caused by prolonged metal-induced oxidative stress. Collectively, these networks validate at the systems level the multi-targeted therapeutic actions of the examined bioactive compounds and support their potential in oxidative toxicity management. The enriched biological processes offer strong evidence for the mechanistic function of BCA, CoQ10, and PHL in attenuating oxidative stress and providing cell protection. The significant enrichment of terms such as response to hydrogen peroxide, superoxide radical removal, and cellular response to oxygen radical is directly related to the antioxidant character of the investigated compounds and their capability to scavenge ROS developed during chromium and arsenic-induced toxicity. Moreover, the enrichment of apoptotic pathways like negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway and muscle cell apoptotic process underscores the protective nature of these compounds in inhibiting ROS-induced cell death. Our results are similar to a study that conducted GO annotation of Rosaceae MIPS genes, revealing their involvement in phytic acid biosynthesis and polyol biosynthesis process^[259]108. These findings are also well supported by the previously discussed protein-protein interaction data, where CASP8, CASP3, and CASP9 were observed to be tightly regulated in apoptosis networks. Our findings align with previous studies in which authors conducted in-depth enrichment analyses of pathways associated with oxidative defense, detoxification, programmed cell death, and several other critical biological processes^[260]66,[261]67,[262]109. These analyses, which provided valuable information, particularly for in-silico studies involving various genes and their associated network functions and biological processes, underscore the importance of such studies in advancing our understanding of complex biological systems. Notably, the compounds’ potential to influence wider stress-response signaling beyond ROS alone, as indicated by terms like response to metal ions, response to chemical stress, and response to nutrient levels, suggests their utility in complex toxicological environments. The systems-level enrichment of these GO terms confirms the multi-target potential of the tested bioactives and supports their candidacy as protective measures against environmental and heavy metal stressors. The improvement in the kidney somatic index, aggregation of metals, and the biochemical changes caused by Cr + As following the administration of (BCA, CoQ10, and PHL) can be related to the antioxidant and metal-chelating activity of the chosen antioxidants. The modulation in the gene expression stood by the biochemical estimation, followed by histological alterations, and our study indicated that the selected therapy might help to counteract the Cr + As-induced nephrotoxicity. In conclusion, BCA, CoQ10, and PHL demonstrated several advantageous traits, including free radical scavenging and anti-oxidation, which are crucial in nephro-protection against Cr + As-induced renal injury. The study’s findings underscore the versatility of BCA, CoQ10, and PHL in demonstrating a variety of nephroprotective pathways. These compounds, with their effective antioxidant and metal-chelating abilities, were found to be responsible for the noted rise in kidney somatic index, decrease in metal buildup, and return of biochemical parameters to normal after treatment. The histopathological results showed structural restoration in renal tissue, and changes in gene expression further validated these therapeutic outcomes. Our findings imply that the chosen antioxidant combination successfully prevents Cr + As-induced nephrotoxicity. In conclusion, BCA, CoQ10, and PHL are potential options for reducing heavy metal-induced kidney damage since they demonstrate a variety of nephroprotective pathways, mainly through the scavenging of free radicals, the strengthening of antioxidant defences, and the reduction of oxidative stress. The selected antioxidant therapy, when used in combination, shows promise as a natural remedy to counteract nephrotoxicity due to chronic exposure to chromium and arsenic in the human population. However, it’s crucial to note some limitations. The short duration of the experiment may not have adequately revealed the long-term toxicological or therapeutic effects of the selected biocompounds. Furthermore, while Swiss albino mice are a commonly used experimental model, they may not fully replicate the complexity of human biochemical and physiological systems. These limitations could limit the direct translational usefulness of the findings. Therefore, it’s important to consider these limitations and the need for further research to confirm and expand on the present findings, including longer treatment periods, different dosing schedules, and humanized experimental models. Conclusion In summary, our study has revealed a potent combination of natural compounds in the fight against renal complications due to exposure to heavy metals. The combination of BCA, CoQ10, and PHL has shown significant benefits in alleviating renal complications against Cr + As-induced cytotoxicity. This is achieved through their anti-oxidation and anti-apoptosis properties in an experimental model. Our findings support the notion that the therapeutic and prophylactic effects of these selected antioxidants are due to their free radicals scavenging property through activation of the SIRT1/Nrf2/KEAP1 signaling pathway and related XMEs (Fig. [263]18). Building on the results of this work, future investigations should examine the long-term and dose-dependent effects of BCA, CoQ10, and PHL in humanized models and cell lines to understand their therapeutic efficacy and safety better. To clarify their precise connections with the Nrf2-KEAP1-ARE signaling axis and other oxidative stress-related pathways, in-depth mechanistic research is required. Furthermore, studies of these compounds’ structure-activity relationship (SAR) may help improve their pharmacological profiles. Additionally, exploring their use in combination with other antioxidants or metal chelators could yield substantial synergistic benefits. Further translational research, including preclinical trials, will provide a comprehensive understanding of the molecular and cellular functioning of BCA, CoQ10, and PHL. This will pave the way for their potential as therapeutic agents in the prevention of oxidative stress-associated renal ailments in the near future. Fig. 18. [264]Fig. 18 [265]Open in a new tab A schematic diagram of different mechanisms of cytoprotection rendered by the selected natural compounds (BCA, CoQ10, PHL) against Cr and As-induced nephrotoxicity. BCA, biochanin‐A; CoQ10, coenzyme Q10; PHL, phloretin. Significance statement BCA, CoQ10, and PHL have been shown to have significant anti-oxidative/free radical scavenging properties. Their modulation of the SIRT1/Nrf2/KEAP1 pathway along with reduced DNA fragmentation, has been observed to preserve renal histology when used as therapeutics against chromium and arsenic-induced nephrotoxicity. The antioxidant characteristics of BCA, CoQ10, and PHL mitigate oxidative damage and enhance the antioxidant response system (biochemical indices) through modulation of the SIRT1/Nrf2/KEAP1 pathway. This combined therapy presents a comprehensive intervention therapy against nephrotoxicity, counteracting the harmful effects of chromium and arsenic while maintaining renal integrity. Our study’s findings opens new avenues for developing focused treatments as supplements to mitigate the detrimental impact of heavy metals on renal physiology in the exposed population. Supplementary Information Below is the link to the electronic supplementary material. [266]Supplementary Material 1^ (916.9KB, pdf) Acknowledgments