Investigation of Thymoquinone's Effects on Bisphenol A-Induced Neurotoxicity, Keap-1/Nrf-2 Signalling, Oxidative Stress, and Behavior in Rats

Summary

This study aimed to investigate the effects of Thymoquinone on oxidative stress, Keap-1/Nrf-2 signalling pathway, and behavior in plasma, the cerebral cortex, and the hippocampus of rats exposed to bisphenol A.

Abstract

Chronic low-dose exposure to bisphenol A (BPA), a widely used environmental endocrine disruptor, has been increasingly associated with oxidative stress-mediated neurotoxicity. Thymoquinone (TQ), a bioactive constituent of Nigella sativa, exhibits potent antioxidant properties; however, its neuroprotective potential against BPA-induced toxicity remains incompletely understood. Given the pervasive nature of BPA exposure, identifying effective and accessible neuroprotective strategies is of growing importance. This study investigated the effects of TQ on oxidative stress, Keap-1/Nrf-2 signaling, behavior, and histopathological alterations in rats exposed to BPA. A total of 24 adult male Wistar Albino rats were randomly assigned to four groups (n = 6/group): control, BPA, BPA + 10 mg/kg TQ (BPA+TQ I), and BPA + 20 mg/kg TQ (BPA+TQ II). BPA (50 µg/kg) and TQ were administered intragastrically for 30 days. BPA exposure induced significant behavioral impairments, oxidative stress, disruption of Keap-1/Nrf-2 signaling, and marked histopathological damage in the hippocampus and cerebral cortex. TQ treatment significantly improved locomotor activity and depression-like behavior, reduced lipid peroxidation and nitric oxide levels, restored glutathione-related antioxidant capacity, and normalized Keap-1 and Nrf-2 expression (p < 0.001). These protective effects were more pronounced at the higher TQ dose. Taken together, these findings demonstrate that thymoquinone mitigates BPA-induced neurotoxicity through modulation of oxidative stress and the Keap-1/Nrf-2 signaling pathway, highlighting its potential as a neuroprotective agent against environmentally relevant BPA exposure.

Introduction

Bisphenol A (BPA) is a synthetic compound widely used in plastics and epoxy resins and is commonly detected in food packaging materials, water systems, medical devices, and household products¹. Continuous environmental exposure has raised concerns regarding its potential neurotoxic effects, particularly under chronic low-dose conditions that reflect real-life human exposure.

Although regulatory agencies such as the U.S. Food and Drug Administration and the U.S. Environmental Protection Agency have defined 50 µg/kg/day as the no-observed-adverse-effect level, emerging experimental and epidemiological evidence indicates that behavioral and cognitive alterations may occur at lower doses. Consequently, the European tolerable daily intake has been reduced to 4 µg/kg/day, emphasizing the need to investigate mechanistic effects of environmentally relevant BPA exposure^1,2.

Oxidative stress is considered a central mechanism underlying BPA-induced neurotoxicity. BPA disrupts cellular redox balance by decreasing antioxidant defenses such as glutathione (GSH) and increasing lipid peroxidation markers, including malondialdehyde (MDA)^3,4. However, the upstream regulatory mechanisms controlling antioxidant responses under BPA exposure remain insufficiently characterized.

The Kelch-like ECH-associated protein 1 (Keap-1)/Nuclear factor erythroid 2-related factor 2 (Nrf-2) pathway is a principal endogenous antioxidant defense mechanism. Under oxidative stress conditions, Nrf-2 dissociates from Keap-1, translocates to the nucleus, and activates antioxidant response element-dependent gene expression⁵. Despite its established role in cellular defense, region-specific modulation of Keap-1/Nrf-2 signaling in the brain during environmentally relevant BPA exposure has not been comprehensively investigated in conjunction with behavioral outcomes.

Thymoquinone (TQ), the major bioactive component of Nigella sativa seed oil, is a lipophilic benzoquinone capable of crossing the blood-brain barrier. TQ exhibits antioxidant, anti-inflammatory, and anti-apoptotic properties, partly through activation of Nrf-2 signaling and enhancement of endogenous antioxidant capacity^5,6. While previous studies have demonstrated the protective effects of TQ in various neurotoxicity models, an integrated methodological approach evaluating oxidative stress markers, Keap-1/Nrf-2 signaling, and behavioral parameters within the same BPA exposure paradigm is lacking^5,6,7.

Here, we describe a reproducible experimental protocol to assess oxidative stress biomarkers, Keap-1/Nrf-2 pathway expression, and behavioral alterations in a rat model of environmentally relevant BPA exposure, and to evaluate the neuroprotective effects of thymoquinone across multiple brain regions.

We hypothesized that low-dose BPA exposure induces behavioral impairments through oxidative stress-mediated dysregulation of the Keap-1/Nrf-2 signaling pathway, and that thymoquinone mitigates these effects by restoring redox balance and activating Nrf-2-dependent antioxidant responses.

Protocol

All experimental procedures were approved by the Institutional Animal Experimentation Local Ethics Committee (Approval No: 68429034/07).

Animals and experimental design
Twenty-four adult male Wistar Albino rats (200-250 g) were obtained from the Experimental Animal Research Laboratory and housed individually under controlled conditions (12 h light/dark cycle, 24 ± 2 °C) with ad libitum access to standard chow and tap water. Animals were acclimatized to laboratory conditions prior to the experiment, which lasted 32 days.

Rats were randomly assigned to experimental groups using a computer-generated simple randomization method to minimize selection bias, with equal numbers of animals allocated to each group (n = 6/group): control, BPA, BPA + 10 mg/kg thymoquinone (BPA + TQ I), and BPA + 20 mg/kg thymoquinone (BPA + TQ II). The sample size was determined by an a priori power analysis using G*Power software (version 3.1). Based on effect sizes reported in previous studies^5,6,7 evaluating BPA-induced neurotoxicity and antioxidant interventions, particularly for biochemical and behavioral outcome measures, a statistical power of 0.80 was assumed for one-way ANOVA. Under these conditions, a minimum of six animals per group was required to detect statistically significant differences among experimental groups.

BPA (50 µg/kg) and TQ (10 or 20 mg/kg) were administered intragastrically by gavage 1x daily for 30 days. All treatments were freshly prepared and administered at a total volume of 1 mL/kg. The BPA dose used in the present study (50 µg/kg/day) was selected based on its designation as a no-observed-adverse-effect level and reference dose by the United States Environmental Protection Agency and the U.S. Food and Drug Administration. This dose has been widely employed in experimental studies to model environmentally relevant BPA exposure and to investigate potential neurobehavioral and biochemical alterations occurring at exposure levels previously considered safe. Importantly, accumulating experimental evidence indicates that adverse neurological and behavioral effects may occur at or below this dose, supporting its relevance for assessing low-dose BPA toxicity^1,2. The selection of TQ doses (10 and 20 mg/kg) was based on previous experimental studies demonstrating its neuroprotective and antioxidant efficacy without inducing toxicity^5,6,7. These doses have been widely used in rodent models of neurotoxicity, oxidative stress, and neuroinflammation, and are well below reported toxicity thresholds for repeated administration. No pilot dose-finding study was conducted; dose selection was guided by literature precedence and established safety profiles to ensure both efficacy and translational relevance.

BPA was administered for 30 consecutive days. Behavioral assessments were conducted on days 31 and 32 following the final exposure. Animals were sacrificed on day 32 after completion of all behavioral tests. This timeline was designed to allow sufficient time for behavioral evaluation while minimizing potential confounding effects of acute handling or procedural stress on subsequent biochemical, molecular, and histopathological analyses. Thymoquinone was administered 1x daily immediately after BPA via the same intragastric route. Behavioral tests were performed following the final BPA and TQ administrations. The experimental timeline is summarized in Figure 1.

Body weight measurement
Body weights were measured using a calibrated digital precision balance (±0.1 g sensitivity). Each rat was weighed individually in the morning before treatment administration, and measurements were recorded on days 1, 10, 20, and 32 under standardized conditions to minimize variability (Figure 2).

Behavioral assessments
All behavioral parameters were scored by a single observer blinded to group allocation to minimize observer bias and inter-observer variability. Animals were habituated to the testing room for at least 30 min prior to behavioral testing to reduce novelty-induced stress.

The combined use of the open field test (OFT) and forced swim test (FST) enables a comprehensive evaluation of BPA-induced neurobehavioral toxicity, as BPA has been shown to alter both emotional reactivity and locomotor activity in rodents^2,3. OFT-derived locomotor outcomes were considered during interpretation of FST immobility to minimize potential confounding effects related to altered motor activity.

FST: Depression-like behavior was assessed using the forced swim test on day 32, preceded by a 15-min pre-test session conducted 24 h earlier. Rats were individually placed in cylindrical tanks (35 cm diameter, 50 cm height) filled with water (30 cm depth, 25 ± 1 °C) and allowed to swim for 5 min. Immobility, swimming, and climbing behaviors were recorded using a video camera and analyzed offline to evaluate behavioral despair⁷.

OFT: Anxiety-like behavior and spontaneous locomotor activity were evaluated using the open field test on day 31. Each rat was placed individually in a white Plexiglas arena (90 cm x 35 cm) divided into 24 equal squares and allowed to explore freely for 5 min. Movements were recorded using a video camera. Time spent in central and peripheral zones and the number of crossings with all four paws were quantified as indices of anxiety-related behavior and locomotion⁸.

Sample collection
On day 32, rats were deeply anesthetized with intramuscular ketamine (45 mg/kg) and xylazine (5 mg/kg). Adequate depth of anesthesia was confirmed by the absence of the pedal withdrawal reflex. Blood was collected via cardiac puncture using a sterile syringe, and approximately 3-5 mL of blood was obtained from each animal. Euthanasia was completed by exsanguination under deep anesthesia. Blood samples were transferred into anticoagulant-containing tubes and centrifuged at 3,000 x g for 10-15 min at 4 °C. The separated plasma was aliquoted and stored at −80 °C until biochemical analysis.

The hippocampus and left cerebral hemisphere were rapidly dissected, weighed, snap-frozen in liquid nitrogen, and stored at −80 °C for subsequent biochemical analyses, while the right cerebral hemisphere was fixed in 10% neutral buffered formalin for histopathological and immunohistochemical evaluations. To ensure methodological consistency and minimize inter-sample variability, the left hemisphere was systematically allocated for biochemical measurements and the right hemisphere for morphological analyses in all animals. Importantly, formalin fixation was strictly limited to tissues designated for histological and immunohistochemical procedures, and no fixed tissue was used for protein-based biochemical assays, thereby preventing fixation-related protein degradation. For biochemical assessments, defined amounts of frozen brain tissue (typically 50-100 mg per assay) were homogenized using assay-specific homogenization buffers and standardized tissue-to-buffer ratios according to the requirements of each biochemical parameter. All analyses were performed using equal tissue amounts within each assay across experimental groups.

Total protein concentration in tissue homogenates was determined using a fluorometric protein assay according to the manufacturer's instructions. All biochemical parameters were measured using standardized and equal protein concentrations within each assay to ensure comparability across samples and experimental groups.

Assessment of oxidative stress parameters
Lipid peroxidation was assessed by measuring MDA levels using the thiobarbituric acid reactive substances (TBARS) method in plasma and tissue samples⁷. Reduced thiol (RSH) and GSH levels were determined spectrophotometrically using 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB)-based assays⁷. Total nitric oxide (NO) levels in plasma and tissue homogenates were measured using the Griess reaction following nitrate reduction⁷. All biochemical parameters are expressed as nmol/g protein or µmol/g tissue, as indicated in the Figure 3 and Figure 4. All biochemical assays were performed in duplicate, and mean values were used for statistical analysis.

In addition to brain tissue analyses, biochemical estimations were performed in blood samples to evaluate the systemic oxidative status induced by BPA exposure. Plasma lipid peroxidation levels were determined by the TBARS method, and results were expressed as MDA equivalents. Reduced sulfhydryl (RSH) levels were measured using DTNB, and total NO levels were assessed using the Griess reaction following nitrate reduction, as previously described⁷. Absorbance values were recorded spectrophotometrically at 532 nm (TBARS), 412 nm (RSH), and 540 nm (NO). All assays were performed in duplicate under standardized conditions.

BPA is a ubiquitous environmental toxicant that enters systemic circulation and exerts multi-organ effects; therefore, circulating oxidative stress markers provide complementary information reflecting overall redox imbalance^1,2. Assessment of blood-based biochemical parameters allows evaluation of the relationship between central and peripheral oxidative alterations and enhances the translational relevance of the findings, as such markers are commonly used in environmental and clinical studies to monitor exposure-related toxicity.

Western blot analysis
Western blot analysis was performed to determine Keap-1 and Nrf-2 protein levels in hippocampal tissue. Approximately 50 mg of tissue was homogenized in 500 µL of RIPA buffer containing a protease inhibitor cocktail. Homogenates were centrifuged at 14,000 x g for 10 min at 4 °C, and the supernatants were collected. Protein concentrations were determined using a fluorometric protein quantification assay according to the manufacturer's instructions, and concentrations were calculated from a standard calibration curve. Equal amounts of protein (30 µg) were mixed with Laemmli buffer containing β-mercaptoethanol, heated at 95 °C for 5 min, separated on 10% SDS-PAGE gels, and transferred onto PVDF membranes at 110 V for 60-90 min. Membranes were blocked with 3% BSA and incubated overnight at 4 °C with primary antibodies against Keap-1 and Nrf-2 (1:3000) and β-actin (1:1000), followed by incubation with HRP-conjugated secondary antibodies. Protein bands were visualized by chemiluminescence, and band intensities were quantified by densitometric analysis and normalized to β-actin.

Histopathological analysis
Brain tissue samples from the right hemisphere were fixed in 10% neutral buffered formalin for 72 h, dehydrated through graded ethanol series, cleared in xylene, and embedded in paraffin according to standard histological procedures⁹. Fresh tissues were reserved for biochemical analyses. Coronal sections (5 µm) were obtained at the hippocampal and entorhinal cortex levels based on a rat brain atlas¹⁰. Four consecutive sections per animal were mounted on glass slides for histological and immunohistochemical evaluations, ensuring consistent section thickness and anatomical localization. Sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) following routine staining protocols¹¹. Histopathological examination was performed under light microscopy by a single trained observer blinded to the experimental groups to minimize bias and eliminate inter-observer variability.

Immunohistochemical analysis
Regions of interest, including the hippocampus and entorhinal cortex, were selected based on their vulnerability to BPA-induced neurotoxicity and their roles in cognitive and emotional regulation^1,2. Immunohistochemical staining for Nrf-2 and Keap-1 was performed on paraffin-embedded brain sections. Sections were deparaffinized in xylene, rehydrated through graded ethanol series, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched prior to incubation.

Sections were incubated overnight at 4 °C with primary antibodies against Nrf-2 (1:100) and Keap-1 (1:200). Detection was performed using a commercially available, ready-to-use polymer-based HRP detection system containing species-specific secondary antibodies conjugated to horseradish peroxidase, according to the manufacturer's instructions. Immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB) as the chromogen, followed by counterstaining with hematoxylin. Sections were dehydrated, cleared, and coverslipped.

Negative control sections were processed in parallel without primary antibody incubation. For quantitative analysis, three non-adjacent sections per animal were evaluated. Images were acquired under identical exposure settings for all groups. Sixteen non-overlapping fields per section were analyzed using ImageJ software. Regions of interest were manually delineated, background staining was subtracted, and immunoreactivity was expressed as mean gray value. The average value per animal was calculated and used for statistical analysis.

Histopathological scoring
Histopathological alterations were evaluated semi-quantitatively on H&E-stained sections. Ten randomly selected, non-overlapping fields per section were examined, and pathological parameters were scored independently. Damage severity in the hippocampus and entorhinal cortex was graded using a four-point scale based on the proportion of affected cells: score 0 (<5%), score 1 (≤ one-third), score 2 (one-third to two-thirds), and score 3 (> two-thirds). Mean scores per animal were used for statistical analysis.

Statistical analysis
Data were analyzed using SPSS software (version 29.0) and are presented as mean ± standard deviation (SD). Sample size was determined based on prior studies and power analysis, indicating that six animals per group provide >80% power to detect biologically relevant differences. Normality of data distribution was assessed using the Shapiro-Wilk test. Homogeneity of variances was evaluated using Levene's test. Group comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Statistical significance was set at p < 0.05.

Results

Body weight
No statistically significant differences in body weight were observed between groups on days 1 and 32 of the study (F(3,20)=2.750, p > 0.05; Figure 2).

Behavioral test results
Open field test analysis revealed that BPA exposure significantly reduced locomotor activity, as indicated by a decreased number of crossings compared to the control group (p < 0.001). Treatment with 10 mg/kg and 2...

Discussion

The present study evaluated the effects of BPA, a structural component of polycarbonate plastics considered safe by the USEPA at a dose of 50 µg/kg^1,2, on oxidative stress, Keap-1/Nrf-2 signaling, behavior, and histopathological alterations in the rat brain. In addition, the potential neuroprotective effects of TQ, a naturally occurring compound with well-documented antioxidant properties, were investigated at two different doses (10 mg/kg and 20 mg/kg)

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5,5'-Dithiobis(2-nitrobenzoic acid)	Thermo Scientific	69-78-3
Benchtop centrifuge NF048	Nüve	43560
Beta_Actin	ThermoFisher Scientific, United States	7D2C10
Bisphenol A	Merck	239658
BSA solution	Proteintech, United States)	66201-1-Ig
Butylated hydroxytoluene	Sigma Aldrich	128-37-0
Dijital Homojenizatör	Daihan Scientific	https://www.artlaboratuvarcih azlari.com/urun/daihan-hg- 15d-dijital-homojenizator-set -a?srsltid=AfmBOorx_az06ulc HpSzKZsuP8-47Za4BkvLOM2 2Xhp00tgwWhJ-86Vc
Hematoxylin–eosin	Bio Optica	W01030708
Hydrochloric acid	Merck	7647-01-0
Image J software program	NIH; Washington, U.S.A.	https://imagej.net/ij/
Immunohistochemical staining kit	Lab VisionTM UltraVisionTM Large Volume Detection System: anti-polyvalent	HRP, TA-125-HL
Leica Autocut 14051956472	Germany	https://www.leicabiosystems. com/histology-equipment/mic rotomes/histocore-autocut/
Light microscope	Nikon, Eclipse	Ni-U, 940728
N-(1-Naphthyl)ethylenediamine	Thermo Scientific	1465-25-4
Nano Microplate Reader	BMG LABTECH	SPECTROstar Nano
phosphate-buffered saline	Merck	P4417
Primary antibodies Keap-1	Proteintech	Cat No. 10503-2-AP, 1:200
Primary antibodies Nrf-2	Proteintech	Cat No. 16396-1-AP, 1/100
Qubit Protein BR Assay Kit	ThermoFisher Scientific-INVITROGEN	Q33211
sodium citrate	Merck	03-04-6132
Sodium Dodecyl Sulfate	Thermo Scientific	151-21-3
sodium hydroxide	Merck	1310-73-2
Sodium nitrate	Merck	7631-99-4
Streptavidin Peroxidase	Thermo Scientific	SHRP248-B
sulfanilamide	Thermo Scientific	63-74-1
thiobarbituric acid	Merck	504-17-6
TQ	CAYMAN	490-91-5
Tricarballylic acid, 99%	Thermo Scientific Chemicals	139360500
Trichloroacetic acid	Thermo Scientific Chemicals	76-03-9
Tris-HCl	Thermo Scientific	1185-53-1
Vanadium(III) chloride	Sigma Aldrich	7718-98-1
West Pico PLUS Chemiluminescent Substrate and the iBright 750	Thermo Fisher Scientific system	https://www.clinxsci.com/product/mini _chemiluminescence_imaging_system ?gad_source=1&gad_campaignid= 16224184202&gbraid=0AAAAAoN6 UOOtoB8UD0deumBAMD8Xi-1l-&g clid=CjwKCAjwgeLHBhBuEiwA L5gNEXtK2CNSLS1GqllMuNgkrFYD aW7yK585njQjFja485Fkwj9lRUEKD BoCgLoQAvD_BwE
Zinc sulfate solution	Merck	7733-02-0