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 ind....

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 u....

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