Introduction

Bisphenol-A (BPA), also known as 2,2-bis(4-hydroxyphenyl) propane, is one of the most prevalent endocrine-disrupting chemicals1,2. Its lipophilic nature, along with its widespread presence in numerous commonly used products3,4 and its detection in various tissues and bodily fluids5, raises significant concerns regarding its potential association with several health conditions, including cancer, cardiovascular diseases, obesity, diabetes, and reproductive disorders6,7,8. Furthermore, BPA’s ability to cross the blood-brain barrier has been linked to neuropsychological and neurobehavioral disturbances9.

BPA can be metabolized into reactive species through the Phase I enzyme system, leading to their accumulation in the mitochondrial membrane. This accumulation can inhibit complex I of the respiratory chain, disrupt electron transport, and increase the production of reactive oxygen species (ROS)10,11,12. By affecting the prooxidant/antioxidant balance in cells, BPA causes antioxidant depletion, induces mitochondrial dysfunction, and triggers changes in cellular signaling pathways, thereby initiating oxidative stress13,14,15. Through mechanisms involving free radicals, BPA may contribute to the development of serious health issues, including neurodegenerative diseases, by damaging protein and lipid structures16. Additionally, prenatal exposure to BPA has been shown to negatively impact children’s behaviors, including social response, working memory, and cognitive abilities17. Our earlier study indicated that BPA could elevate ROS levels and induce sperm dysfunction by decreasing enzymatic and non-enzymatic antioxidant levels in rats18. However, the effects of BPA on the central and peripheral nervous systems remain uncertain, highlighting the need to investigate BPA’s potential to induce oxidative stress-mediated neurotoxicity in these tissues.

Antioxidant systems play a crucial role in protecting neuronal cells from oxidative stress in both the peripheral nervous system (PNS) and central nervous system (CNS). In these areas, where extracellular space is limited and distances to neighboring cells are minimal, these systems neutralize reactive oxygen species and maintain gene regulation, preserving the integrity of the cellular environment. This protection is vital for combating oxidative stress and neurodegenerative diseases19. In recent years, natural antioxidants have emerged as a more appealing option for treating neurological disorders due to their comparatively lower toxicity, availability, and cost20,21,22.

Derived from a medicinally potent plant with a rich traditional history, Nigella sativa (NS) and its seed oil (NSO) have been documented to exhibit a broad range of therapeutic properties, including antimicrobial, anxiolytic, anti-inflammatory, anticancer, antitussive, and antioxidant effects23,24. The preventive impact of NSO includes the inhibition of lipid peroxidation, enhancement of total thiol content and glutathione levels, scavenging of free radicals, and augmentation of enzymatic antioxidant activities, as well as the inhibition of NF-κB activity and suppression of cyclooxygenase and lipoxygenase through various mechanisms25. Furthermore, both NS and NSO, particularly the active compound thymoquinone (TQ), have demonstrated a wide array of therapeutic benefits in both in-vivo and in-vitro studies. Preclinical toxicity studies, following the Organization for Economic Co-operation and Development (OECD) guidelines, suggest a safe dosage of NSO in humans should not exceed 900 mg per adult per day or 50 mg of TQ per adult per day26. These findings indicate promising outcomes in protecting against various neurodegenerative diseases27,28,29,30,31,32.

The data presented in this study support the hypothesis that NSO containing TQ may serve as a protective agent against BPA-induced neurodegeneration. However, given the limited scientific literature on the neuropharmacological effects of NSO on BPA-related neurodegeneration, our study aims to address this knowledge gap. Specifically, this research seeks not only to elucidate the neurotoxic effects of BPA-induced oxidative stress on both the central and peripheral nervous systems but also to demonstrate the neuroprotective properties of NSO against BPA-induced toxicity at enzymatic, molecular, and histopathological levels.

Results

The normalizing effect of NSO on lipid peroxidation induced by BPA and antioxidant enzyme levels

Figure 1 illustrates the levels of malondialdehyde (MDA) and reduced glutathione (GSH) in tissue samples from the brain and sciatic nerve. In the BPA group, MDA levels were significantly elevated in both tissues compared to the control group (p < 0.05). In contrast, GSH levels were markedly reduced in the BPA group (p < 0.05). Notably, the co-administration of Nigella Sativa oil (BPA + NSO), assessed for its therapeutic potential, led to a significant reduction in MDA levels and a concomitant increase in GSH levels in comparison to the BPA group (p < 0.05) (Fig. 1).

Fig. 1
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Brain/sciatic nerve and tissue oxidant and antioxidant marker levels of rats poisoned with control, NSO (5 ml/kg bwt/day) and/or Bisphenol A (BPA, 100 mg/kg bwt/day). Data are presented as mena + SD. ‘**’ indicates a statistically significant difference compared to the control group (p < 0.05), ‘##’ indicates a statistically significant difference compared to the BPA group (p < 0.05). ‘ns’ indicates a statistically insignificant difference compared to the control group (p > 0.05). MDA; malondialdehyde, GSH; glutathione.

The therapeutic effect of NSO on brain and sciatic nerve NRF2, NF-κB, Capase-3, and NR4a2 mRNA expression levels ınduced by BPA in rats

Figures 2 and 3 present the RT-PCR analyses conducted to examine the anti-inflammatory, antioxidant, and anti-apoptotic effects of Nigella Sativa oil (NSO) on Bisphenol A (BPA)-induced damage in the brain and sciatic nerve. The results demonstrate that BPA exposure significantly increased mRNA expression levels of Nrf2, NF-κB, and caspase-3, while decreasing NR4A2 levels in both brain and sciatic nerve tissues compared to the control group (p < 0.05).

In contrast, the co-administration of NSO with BPA produced significant restorative effects on the mRNA levels of caspase-3 and NF-κB in the brain, with values approaching those of the control group (p < 0.05). Additionally, in the BPA + NSO group, Nrf2 mRNA levels decreased, and NR4A2 levels increased, in comparison to the BPA group (p < 0.05) (Fig. 2).

Fig. 2
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Gene expression levels of relative Nrf2, NF-κB, NR4A2 and Caspase 3 mRNA transcripts in the brain of control, NSO (5 ml/kg bwt/day) and/or Bisphenol A (BPA, 100 mg/kg bwt/day) poisoned rats. Data are presented as mena + SD. ‘**’ indicates a statistically significant difference compared to the control group (p < 0.05). ‘##’ indicates a statistically significant difference compared to the BPA group (p < 0.05). ‘ns’ indicates a statistically insignificant difference compared with the control group (p > 0.05).

In the BPA + NSO group, the mRNA expression levels of NR4A2, Nrf2, caspase-3, and NF-κB in the sciatic nerve were significantly improved compared to the group treated with BPA alone (p < 0.05). Additionally, the levels of NR4A2, Nrf2, and NF-κB in the BPA + NSO group closely resembled those of the control group (Fig. 3). These results underscore the potential of NSO to mitigate and reverse the neuropathological effects induced by BPA.

Fig. 3
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Gene expression levels of the sciatic nerve relative Nrf2, NF-κB, NR4A2 and Caspase 3 mRNA transcripts of control, NSO (5 mg/kg bwt/day) and/or Bisphenol A (BPA, 100 mg/kg bwt/day) poisoned rats. Data are presented as mena + SD. ‘**’ indicates a statistically significant difference compared to the control group (p < 0.05). ‘##’ indicates a statistically significant difference compared to the BPA group (p < 0.05). ‘ns’ indicates a statistically insignificant difference compared with the control group (p > 0.05).

Histopathological findings

The results of the histopathological and statistical analyses are summarized in Table 1. In the brain tissue of animals from the healthy control group, only minor variations within physiological limits were observed, including mild to moderate hemorrhage in the meninges and submeningeal region in two animals and slight hyperemia in blood vessels. In contrast, the BPA group exhibited pronounced pathological changes, such as hydropic degeneration of neurons, single-cell necrosis (apoptosis), increased glial cell numbers, neuronophagy, perineuronal/neuropil edema, endothelial cell swelling in blood vessels, hyperemia, hemorrhage in meninges and neuropil tissue, and infiltration of mononuclear cells (MNC) (Fig. 4).

No significant differences were observed in the NSO group compared to the healthy control group. However, the BPA + NSO group showed significant improvement in neuronal degeneration/necrosis, neuronophagy, gliosis, MNC infiltration, hyperemia, edema, and endothelial cell swelling (Fig. 5).

Table 1 The mean values of the histopathological findings observed in the cerebral cortex, hippocampus, cerebellum and brain stem.
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Fig. 4
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Histopathological findings, BPA group, HE. A. Hemorrhage (black arrow) and oedema (blue arrow) in neuropil tissue, Scale bar: 100 μm; B. Neuronal degeneration and perineuronal oedema (yellow arrow) and neuronophagy (red arrow), Scale bar: 50 μm, C. Central chromatolysis (green arrow) and neuronophagy (red arrow), Scale bar: 100 μm, D. Hemorrhage and inflammation (black arrow) in meninges, Scale bar: 100 μm.

Fig. 5
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Histopathological findings, all groups, HE. A. Control group; B. BPA group, neuronal degeneration and perineuronal oedema (yellow arrow), neuronophagy (red arrow) and central chromatolysis (green arrow); C. NSO group, neuronophagy (red arrow); D. BPA + NSO group, neuronal degeneration and perineuronal oedema (yellow arrow), neuronophagy (red arrow), Scale bars: 50 μm.

Immunohistochemical findings

Table 2 provides the results of immunohistochemical staining. These findings determined that BPA notably increased GFAP expression in the cerebral cortex and hippocampal regions. However, in the BPA + NSO group, this increase was notably mitigated (Fig. 6). No significant differences were observed among the groups in the cerebellum and brainstem.

Table 2 Immunohistochemical staining results.
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Fig. 6
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Immunohistochemical findings. A: Mild GFAP immunopositivity, Control group, Scale bar: 100 μm; B: Severe GFAP immunopositivity, BPA group, Scale bar: 200 μm; C: Mild GFAP immunopositivity, NSO group, Scale bar: 100 μm; D: Moderate GFAP immunopositivity, BPA + NSO group, Scale bar: 200 μm.

Multivariate, correlation and clustering analyses

Multivariate, correlation, and clustering analyses were conducted following the methodology outlined by Farag, et al.33. Using a combination of hierarchical cluster analysis, supervised PCA, and unsupervised PLS-DA, clear differentiation among the experimental groups was achieved (Figs. 7 and 8). The BPA model group exhibited a distinct separation from both the control and NSO groups, positioned further to the left, confirming the successful establishment of the BPA-induced animal model. Notably, the NSO-treated group closely aligned with the control group, indicating their similarity. Of particular significance is the proximity of the BPA + NSO group to the control group, highlighting the neuropharmacological efficacy of NSO in mitigating the neurodegenerative effects induced by BPA.

Fig. 7
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Heatmap summarises the effect of NSO on biomarkers studied in BPA-exposed rats. Coloured boxes indicate the effect induced by up-regulation (red) or down-regulation (blue).

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‘a’ Principal component analysis and ‘b’ partial least square discriminant analysis score plots among selected components of the biomarkers investigated during the study of the effects of NSO on BPA-exposed rats.

In the sPLS-DA model, BrainGFAPHippo, BrainGFAPCorte, and BrainMDA were identified as the biomarkers with the highest Variable Importance in Projection (VIP) scores, emphasizing their critical role in accurately distinguishing between the experimental groups (Fig. 9). Furthermore, Pearson’s correlation analysis (r) was performed to evaluate the relationships between the various biomarkers, with both positive and negative correlations visually depicted (Supplementary Fig. 1).

Fig. 9
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Variable importance projection (VIP) score plot detecting significant biomarkers identified by sPLSDA during the study of the effects of NSO on BPA-exposed rats. The coloured boxes on the right indicate each experimental group’s relative regulation of the respective marker.

Discussion

BPA can induce detrimental effects on cells, organelles, and organs through interactions with various cell surface receptors. This interaction may generate free radicals, leading to cellular toxicity, structural alterations, DNA damage, mitochondrial dysfunction, centriole duplication, and abnormal modifications in certain cellular signaling pathways34. As an environmental pollutant with established harmful effects, BPA can cause multiple neuropsychological and neurobehavioral disorders by crossing the blood-brain barrier9. This prompted the researchers in the current study to investigate the potential impact of Nigella Sativa Oil (NSO), known for its neuroprotective properties, with the aim of elucidating the molecular mechanisms underlying BPA-induced neurotoxicity—an area with limited information—and to develop practical solutions.

BPA can undergo oxidation, transforming into catechol and subsequently into O-quinone. This process results in the formation of reactive oxygen species (ROS) within nervous system cells, achieved by the rapid conversion of superoxide radicals into hydrogen peroxide and highly reactive hydroxyl radicals35. This study demonstrated that BPA administered at a dose of 100 mg/kg body weight for 30 days significantly increased malondialdehyde (MDA) levels in the brain and sciatic nerve while decreasing glutathione (GSH) content due to antioxidant depletion. The observed decrease in GSH and increase in MDA levels indicated a disrupted oxidant/antioxidant balance, leading to oxidative stress in both brain and sciatic nerve tissues. Histopathological findings supported this, revealing neurodegenerative changes such as hydropic degeneration in neurons, single-cell necrosis (apoptosis), increased glial cell numbers, neuronophagy, and perineuronal edema in the BPA group.

Immunohistochemical analysis also demonstrated that BPA exposure led to an increase in glial fibrillary acidic protein (GFAP) expression, an important marker of neurodegeneration, particularly in the cerebral cortex and hippocampus. Furthermore, BPA exposure was associated with elevated caspase-3 mRNA transcript levels in the examined tissues. Apoptosis is recognized as a significant mechanism underlying BPA-induced neurodegeneration36,37. Collectively, these results suggest that BPA exposure triggers oxidative stress by increasing MDA levels and depleting GSH content in both the CNS and PNS, leading to apoptosis and resultant neurodegeneration14,38,39,40,41,42.

Cellular responses to inflammation and stress, as well as the redox balance within cells, are regulated through the Nrf2 and NF-κB pathways, ensuring proper cellular function43. Many neurotoxic substances can enhance neurotoxicity and trigger neurodegenerative diseases by activating the Nrf2/NF-κB pathways44,45. Nrf2 is a transcription factor that regulates cellular responses to oxidative stress46. Under normal conditions, the protein Keap1 retains Nrf2 in the cytoplasm, facilitating its proteasomal degradation and maintaining low levels of Nrf247. In the presence of oxidative stress, Nrf2 dissociates from Keap1, translocating to the nucleus where it binds to the antioxidant response element (ARE). This process facilitates the transcription of endogenous antioxidative genes, ensuring a cellular response to oxidative challenges46,48. During oxidative stress, NF-κB is released due to the phosphorylation of IκB, subsequently translocating to the nucleus and promoting the transcription of pro-inflammatory mediators49. In our study, the significant increase in Nrf2 and NF-κB gene expressions in both brain and sciatic nerve tissues indicates that BPA triggers oxidative stress and inflammation. The elevation of Nrf2 and NF-κB levels correlates with BPA-induced GSH depletion and lipid peroxidation. BPA may activate the apoptotic response through ROS accumulation, where Nrf2 cannot counteract cytotoxicity and ROS storage, and exposure to high doses may lead to overexpression of Nrf250,51.

Moreover, Nrf2 is known to regulate the sensitivity of cell death receptor signaling via intracellular glutathione levels in vivo, and this signaling is inhibited by Nrf2 52. The increase in cell death signaling results from an inadequate stress response and serves as a protective mechanism to eliminate damaged cells52,53. Our study observed that BPA increased the accumulation of MDA from lipid peroxidation while depleting both enzymatic and non-enzymatic antioxidants, acting as a stress factor that leads to ROS accumulation and stimulating the inflammatory response through the NF-κB signaling pathway54,55. Histopathological findings of neuroinflammation, such as hyperemia, edema, gliosis, and perivascular infiltration, were more pronounced in the BPA group.

Notably, decreased mRNA transcript levels of NR4A2—essential for protection against oxidative stress and inflammation related to neurodegenerative disorders—were observed in both tissues. Previous studies have delineated the critical roles of NR4A2 across various biological domains, including oncogenesis, inflammation, metabolism, immunity, and autophagy56,57,58,59. A decline in NR4A2 expression has been linked to heightened endogenous apoptosis in HeLa cells due to increased ROS levels and activation of endoplasmic reticulum stress60. Recent evidence has highlighted the association of NR4A2, widely expressed in the CNS, with cerebral nerve development and neurological diseases. NR4A2 regulates the expression of various genes related to neuronal growth, differentiation, and survival56. While the effects of BPA on cell growth and proliferation via estrogenic receptors are acknowledged, the precise mechanisms underlying the initiation of neuronal apoptosis remain incompletely understood. The data presented in this study suggest that BPA induces neurodegeneration through a dual mechanism: triggering neuronal apoptosis by reducing NR4A2 expression and directly causing oxidative stress and antioxidant blockade.

Given the rich history of plant use in traditional medicine, alongside their accessibility, low cost, and general acceptance as safe and biologically relevant alternatives to synthetic compounds, there is growing interest in exploring the therapeutic potential of various plant species61. Numerous studies have consistently highlighted the therapeutic potential of NSO and its active component, thymoquinone (TQ), in addressing a broad spectrum of diseases, including chronic non-communicable and infectious diseases62. In the present study, simultaneous administration of NSO with BPA resulted in a significant decrease in both brain and sciatic nerve MDA levels and an increase in GSH activity, suggesting that NSO can alleviate oxidative stress by restoring the disrupted oxidant/antioxidant balance. NSO also decreased the gene expression levels of Nrf2, NF-κB, and caspase-3 while increasing NR4A2 expression levels in both tissues. This suggests that NSO is neuroprotective against BPA-induced neurodegeneration through its antioxidant, anti-inflammatory, and anti-apoptotic properties. Histopathological examinations further demonstrated the neuroprotective activity of NSO, showing that it attenuated BPA-induced neurodegeneration, supported by decreased GFAP immunoreactivity.

Several studies suggest that the neuroprotective activity of NSO is attributable to the antioxidant, anti-inflammatory, and anti-apoptotic properties of thymoquinone63,64,65,66. Lotfi, et al.63 demonstrated the neuroprotective effects of TQ against nonylphenol (NP)-induced neurotoxicity, revealing that TQ mitigates oxidative damage and neuroinflammation, thereby countering NP-induced memory deficits and neurotoxicity. Dong, et al.64 showed that TQ mitigates progressive dopaminergic neuropathology in vivo and in vitro by activating the Nrf2/ARE signaling cascade and reducing oxidative stress. Kanter65 found that TQ, particularly in conjunction with NS treatment, led to morphological improvements in neurodegeneration within the hippocampus following chronic toluene exposure in rats. In addition, Ullah, et al.66 reported that TQ may provide neuroprotection by inhibiting the apoptotic cascade through modulation and stabilization of mitochondrial membrane potential. Consistent with these findings, the 21.5 mg/g TQ present in the NSO used in our study is believed to exhibit potential neuroprotective effects in both tissues due to its radical scavenging activity. Furthermore, the present study demonstrated that NSO-induced NR4A2 expression contributed to the reduction of intracellular oxidative stress and apoptosis. However, further research is necessary to elucidate the receptor-level interactions and subsequent intracellular signaling pathways to clarify the relationship between the neuroprotective activity of NSO or its active component TQ and NR4A2.

In conclusion, BPA administered at a dose of 100 mg/kg body weight caused lipid peroxidation and decreased antioxidant levels in both the CNS and PNS. It resulted in a significant increase in Nrf2 and NF-κB mRNA transcript levels due to oxidative stress, alongside a decrease in neuroprotective NR4A2 gene expression, leading to neuropathological findings. This study provides biochemical, molecular, and histopathological evidence of the protective effects of NSO, a potent antioxidant, against BPA-induced neurodegeneration. Notably, NSO activates NR4A2 expression, which is critical for mitigating neuroinflammation and neuronal cell death, despite the stress induced by BPA. While additional research is needed to determine whether NSO activates or modulates NR4A2 in neurodegenerative and neurobehavioral disorders, the present study offers promising evidence that NSO may serve as a novel therapeutic agent for treating neurodegenerative disorders.

Methods

Ethics statements

All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals67. Experimental procedures were conducted at the Selcuk University Experimental Application and Research Center in Turkey. Ethical approval was obtained from the Ethics Committee of Selcuk University Faculty of Veterinary Medicine Animal Production and Research Center, with the protocol receiving reference number 2022/34. All research activities adhered strictly to the principles set forth by the European Economic Community Directives (86/609/EEC and 2010/63/EU) regarding animal welfare. The study is in accordance with the ARRIVE guidelines.

Animals and experimental protocol

The investigation spanned a duration of 30 days and involved a cohort of 36 robust adult Wistar Albino rats, each weighing between 400 and 450 g. During the study, the rats were housed in plastic cages, maintained on a 12/12 light-dark cycle, and kept in an environment with a temperature of 22 ± 2 °C and a humidity level of 50%±10%. After a seven-day acclimatization period, the rats were carefully divided into four distinct groups based on comparable mean body weights. These groups were designated as control, NSO, BPA, and BPA + NSO, reflecting the experimental interventions under examination.

  • Group 1 (Control, n = 6): Throughout the trial period, standard rat feed and unrestricted access to drinking water were consistently provided.

  • Group 2 (BPA, n = 10): Throughout the trial period, standard rat feed and unrestricted access to drinking water were consistently provided. Bisphenol A (BPA) at 100 mg/kg was administered via gavage throughout the trial68.

  • Group 3 (NSO, n = 10): Throughout the trial period, standard rat feed and unrestricted access to drinking water were consistently provided. NSO containing 21.5 mg/g TQ was administered via gavage at 5 ml/kg throughout the trial. NSO was given at a dosage corresponding to 1.25% of the daily food ratio69.

  • Group 4 (BPA + NSO, n = 10): Throughout the trial period, standard rat feed and unrestricted access to drinking water were consistently provided. A 100 mg/kg BPA dosage was administered, followed by 5 ml/kg NSO 45 min later. According to a previous study, the maximum plasma concentration of BPA (400 µg/kg, a radioactive form of BPA) was reached 20 min after oral administration70. Therefore, in the current study, TQ and NSO were administered 30 min after BPA gavage, when BPA’s plasma concentration was expected to reach its peak.

Botalife (Isparta, Turkey) provided the NSO, and Sigma Chemical Company (St. Louis, Mo, USA) acquired the BPA. The company that furnished the product conducted the HPLC method, which confirmed the thymoquinone (TQ) content in the NSO as 21.5 mg/g.

Sample collection and preparation

Twenty-four hours after the final administration, the rats were euthanized under general anesthesia using a combination of ketamine (90 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally. Euthanasia was performed via cervical dislocation. Following this procedure, the brains were swiftly extracted and divided into right and left hemispheres after careful rinsing with isotonic saline solution. Similarly, the sciatic nerves were dissected using appropriate anatomical techniques. Specimens from the right cerebral hemisphere and sciatic nerve were rapidly frozen in liquid nitrogen and stored at -80 °C until RNA extraction and antioxidant level assessments were conducted. Samples from the left cerebral hemisphere were preserved in a 10% formalin solution for subsequent histopathological and immunohistochemical evaluations.

Assessment of oxidative/antioxidant indices

The right cerebral hemisphere and sciatic nerve tissues were rinsed with cold isotonic saline, then sectioned and homogenized in ice-cold sodium-potassium phosphate buffer (1.15% KCl, 0.01 M, pH 7.4) at a concentration of 10% (w/v). Following homogenization, the samples were centrifuged at 5000 rpm for 10 min at 4 °C, and the resulting supernatants were aliquoted and stored at -80 °C for subsequent analyses related to lipid peroxidation and non-enzymatic antioxidant parameters38.

Malondialdehyde (MDA) levels were quantified using the ELISA method, following the procedure by Ohkawa, et al.71. Reduced glutathione (GSH) concentrations were determined with the ELISA method, as per the protocol by Beutler, et al.72. Spectrophotometric measurements at 450 nm were performed using an ELx800 instrument (Bio-Tek Instruments, Winooski, VT, USA), and OD values were correlated with standard concentrations through separate standard curves generated with Microsoft Excel using linear regression equations.

Quantitative real Time-PCR (qRT-PCR) and RNA isolation

Gene expression analysis followed established procedures 70. Total RNA was extracted from snap-frozen brain and sciatic nerve samples using the SanPrep Column microRNA Miniprep Kit (BIO BASIC), adhering to the manufacturer’s instructions. For cDNA template synthesis, 2 µg of the isolated RNA aliquot underwent reverse transcription with the OneScript® Plus cDNA Synthesis Kit (ABM) using an oligo dT primer. Fluorescent real-time quantitative PCR (RT-qPCR) was employed to measure the mRNA levels of Nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), Nuclear Factor kappa B (NF-κB), Nuclear receptor subfamily 4 group A member 2 (NR4A2), and Caspase-3. BlasTaq™ 2X qPCR MasterMix (ABM) and the primer sequences summarized in Table 3 were utilized for PCR reactions conducted in triplicate on a LightCycler® 96 System (Roche, Switzerland). The thermal program included an initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation (95 °C/10 s), annealing (56 °C/15 s), and extension (72 °C/10 s). Relative gene expression was calculated using the comparative cycle threshold (2-ΔΔCT) method, normalizing target mRNA Ct values to those of ß-actinin as an internal control. Additionally, a melting curve analysis was performed to confirm the amplification of a single PCR product, ensuring the robustness of the results.

Table 3 Primer sequence.
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Histopathological and immunohistochemical analysis

After systemic necropsy of euthanized rats, their brains (hemispheres, cerebellum, brainstem) were dissected for macroscopic examination. The right hemisphere, divided by a sagittal section passing through the midline of the hemispheres, cerebellum, and brainstem, was immersed in a 10% buffered formaldehyde solution for fixation over 48 h. Following fixation, the brain samples were washed in running tap water and placed in a tissue processor (Leica TP1080) for routine pathological tissue processing. The processed samples were then sectioned to a thickness of 5 μm using a microtome (Leica RM-2125 RT) and mounted onto positively charged slides. All sections were initially stained using the Hematoxylin and Eosin (HE) staining method73. Additionally, sections were immunohistochemically stained for Glial Fibrillary Acidic Protein (GFAP, 1:1000, ab59348, Abcam, USA) using a fully automated immunohistochemistry staining system (Bondmax) and kits (Leica DS9800) as per the previously reported method74. All samples were examined using a binocular-head light microscope (Olympus BX51, Tokyo, Japan). Histopathologically, the cerebral cortex, hippocampus, cerebellum and brainstem sections of the brain were evaluated in five different fields at 40x objective magnification for neuronal degeneration/necrosis, gliosis, neuronophagia, edema, hemorrhage, hyperemia, mononuclear cell infiltration and endothelial cell swelling, and were scored as mild (1), moderate (2), severe (3) or very severe (4)74,75. Furthermore, the scoring of GFAP immunoreactivity was conducted separately for the prevalence and intensity of immunostaining in the cerebral cortex, hippocampus, cerebellum, and brainstem, in accordance with the previously reported methodology74.

Statistical analyses

The normal distribution of the acquired numerical values was assessed using the Shapiro-Wilk test, while Levene’s test was utilized to evaluate the homogeneity of variances. Data exhibiting a normal distribution were analyzed using one-way ANOVA followed by post hoc Duncan tests. Results are expressed as mean ± standard error in the tables, with a significance level of p < 0.05 considered statistically significant. Significance markers were used as follows: (**) for p < 0.05, indicating notable differences compared to the control group, and (##) for p < 0.05, denoting significant differences compared to the BPA group. Values marked as (ns) for p > 0.05 indicated no significant differences compared to the control group, while (ns*) indicated no significant differences compared to the BPA group. The F values from the one-way ANOVA analysis were reported for each parameter to provide a detailed measure of variance between the groups (Supplementary Table 1).

All biological outcomes underwent processing that included quantile normalization, log transformation, and Pareto scaling methods for multivariate data analysis, correlation, and clustering analysis33. The analysis involved unsupervised supervised Partial Least Squares-Discriminant Analysis (PLS-DA) and Principal Component Analysis (PCA) methods33,76. Additionally, Pearson’s R correlations were applied for univariate correlation analysis, and hierarchical clustering utilized Euclidean distance measurement along with Ward’s algorithm33. The creation of all figures was conducted using MetaboAnalyst 4.0. and GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, California, USA).