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Abstract

Polychlorinated biphenyls (PCBs) comprise a ubiquitous class of toxic substances associated with carcinogenic and tumor-promoting effects as well as neurotoxic properties. Reactive oxygen species, which is produced from PCBs, alters blood–brain barrier (BBB) integrity, which is paralleled by cytoskeletal rearrangements and redistribution and disappearance of tight junction proteins (TJPs) like claudin-5 and occludin. Quercetin, a potent antioxidant present in onion and other vegetables, appears to protect brain cells against oxidative stress, a tissue-damaging process associated with Alzheimer’s and other neurodegenerative disorders. The aim of this study is to analyze the role of quercetin on oxidative stress markers and transcription of transmembrane and cytoplasmic accessory TJPs on cerebrum, cerebellum and hippocampus of female rats exposed to PCBs. Rats were divided into the following four groups. Group I: received only vehicle (corn oil) intraperitoneally (i.p.); group II: received Aroclor 1254 at a dose of 2 mg/kg body weight (bwt)/day (i.p); group III: received Aroclor 1254 (i.p.) and simultaneously quercetin 50 mg/kg bwt/day through gavage and group IV: received quercetin alone gavage. From the experiment, the levels of hydrogen peroxide, lipid peroxidation and thiobarbituric acid reactive substances were observed to increase significantly in cerebrum, cerebellum and hippocampus as 50%, 25% and 20%, respectively, after exposure to PCB, and the messenger RNA expression of TJP in rats exposed to PCBs is decreased and is retrieved to the normal level simultaneously in quercetin-treated rats. Hence, quercetin can be used as a preventive medicine to PCBs exposure and prevents neurodegenerative disorders.

Keywords

Blood–brain barrier tight junction proteins polychlorinated biphenyls quercetin

Introduction

Polychlorinated biphenyls (PCBs) are persistent environmental toxicants associated with numerous adverse health effects.¹ PCBs are widely used in electrical industry, as coolants for transformers and capacitors.² PCBs are lipophilic and resistant to biological decomposition and can accumulate in higher tropic levels through the food chain.³ Most of the systemic toxic and biological effects of PCBs are mediated by the aryl hydrocarbon receptor (AhR). Aroclor 1254 is a technical mixture containing almost 70 different PCB isomers and congeners with a molecular mass ranging from 188 to 430 Da,⁴ which has been widely utilized in studying PCB toxicity. As far as its neurotoxicity is concerned, Aroclor 1254 has been shown to produce neurochemical alterations in several experimental settings⁵ as well as behavioral changes in learning, memory, motor activity and sexual behavior.^6,7 Developmental exposure to PCB mixture or congeners altered the motor activity, neurological development and cognitive function in laboratory animals.⁸ Our earlier studies suggest that the PCB increases the reactive oxygen species (ROS) level and decreases the activities of antioxidant enzymes, functional markers like creatine kinase, acetyl choline esterase and membrane bound ATPases in selected rat brain regions.^9

–13

Homeostasis of the central nervous system (CNS) microenvironment is essential for normal functions of the brain and is maintained by the blood–brain barrier (BBB). The BBB is mainly formed by highly specialized endothelial cells (ECs), and it protects the brain from blood-borne substances. At the same time, the BBB ensures the supply of nutrients to the brain by specific transport systems.¹⁴ The BBB acts as a physical and metabolic barrier because a complex tight junction (TJ) system between the adjacent ECs restricts most paracellular movement of ions and solutes across the brain endothelium.¹⁵ TJs of the BBB are composed of an intricate combination of at least three integral membrane proteins (claudins (Cldn), junction adhesion molecules (JAMs) and occludin (Ocln)) and cytoplasmic accessory proteins, such as zonula occludens (ZO)-1, ZO-2 and ALL-1 fusion partner at chromosome-6 (AF6).

ZO-1 was the first TJ-associated protein identified and characterized among cytosolic accessory proteins of TJ.¹⁶ The ZO proteins belong to the family of membrane-associated guanylate kinases (MAGUK), which possess a distinct modular organization and associate peripherally with the cellular membranes. ZO-1 is essential for TJ assembly, and it forms a scaffold complex with other cytosolic accessory proteins, such as ZO-2 and AF6, to anchor integral membrane proteins to actin cytoskeleton.^17,18 Disruption of ZO-1 at TJ has been observed in response to bacterial toxins,¹⁹ drugs,²⁰ growth factors,²¹ cytokines²² and hypoxia²³ and correlated with increased permeability and/or decreased electrical resistance. In addition, ZO proteins are believed to form a complex for transcription factors and signaling proteins involved in the regulation of cell proliferation and differentiation.^24,25

Relatively less information is available on the role of ZO-2 and AF6 in the regulation of integrity of the brain endothelium. ZO-2 shares three-defined core regions of MAGUK family proteins with ZO-1, which are a Src homology 3 (SH3) domain, a guanylate cyclase and a PDZ (PDZ is an acronym combining the first letters of three proteins – post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1) – which were first discovered to share the domain) domain. SH3-domains bind with signaling proteins and cytoskeletal elements. Guanylate cyclase domains are involved in the adenosine triphosphate (ATP)-dependent transformation of guanosine monophosphate (GMP) to guanosine diphosphate (GDP), and the PDZ-domains mediate specific binding to carboxy-terminal cytoplasmic ends of transmembrane proteins.¹⁸ ZO-2 acts as a signaling molecule to communicate the state of cell–cell contact of the TJ.²⁶ It appears that ZO-2 can modulate these interactions by its influence on the transcription factors c-Jun, c-Fos and C/EBP (CCAAT-enhancer binding proteins).²⁷ ZO-2 expression decreases in response to several pathological conditions, including amyloid beta treatment, hypoxia,²³ cerebral infarction in spontaneously hypertensive rats²⁸ and exposure to HIV proteins, such as gp120²⁹ or Tat.³⁰

AF6 is a multidomain actin-binding protein that serves as a scaffold protein between transmembrane proteins and the actin cytoskeleton.³¹ It interacts with ZO-1, nectin, Eph receptors and the actin-regulatory protein profiling.^31,32 It binds to Ras-like small GTPases and is suggested to be an effector for both Ras and Raf. It has been shown that the activation of Ras can result from the interaction of AF6 with ZO-1.³² AF6 may regulate the adhesion of leukocytes to extracellular matrix. Indeed, overexpression of AF6 in Jurkat cells increased its adhesion to fibronectin. In contrast, knockdown of AF6 in T-cells results in an enhancement of cell adhesion.³³

Exposure to PCBs can contribute to the induction of neurological disorders including morphological changes in neurons,³⁴ brain tumor promoting effects³⁵ and the developmental defects of the nervous system.³⁶ However, the mechanisms by which PCBs cause these neurotoxic effects are not fully understood. BBB integrity is paralleled by cytoskeleton rearrangements, redistribution and disappearance of tight junction proteins (TJPs), such as claudin-5 (Cldn5) and Ocln. ROS induces enhanced Ca²⁺ mobilization and inositol (1,4,5)-triphosphate formation in brain ECs via phospholipase C.³⁷ The organization of TJPs, Ocln and ZO-1, is altered by exogenous ROS in brain ECs.³⁸ ROS selectively activates signaling cascades involving Ras homolog gene family; member A (RhoA) is a small GTPase protein known to regulate the actin cytoskeleton in the formation of stress fibers. In humans, it is encoded by the gene RhoA, phosphoinositide 3-OH (PI3) kinase and protein kinase B (PKB/Akt), leading to the rearrangements of the actin cytoskeleton and spatial redistribution, disappearance of Ocln and Cldn5, inducing altered BBB integrity. ROS disrupts the integrity of TJ, by transient activation of the PI3 kinase and PKB pathway via RhoA.³⁹

ROS may activate protein tyrosine kinases in brain ECs, which in turn may lead to tyrosine phosphorylation of TJ proteins.⁴⁰ ROS selectively activates signaling cascades involving RhoA, PI3 kinase and PKB/Akt leading to the rearrangements of actin cytoskeleton and spatial redistribution and disappearance of Ocln and Cldn5, inducing altered BBB integrity. ROS induces PKB phosphorylation through RhoA activation.³⁹ The family of Ras-related small GTP-binding proteins comprises RhoA, Rac1 and Cdc42, which are the regulators of the actin cytoskeleton, and it has been described that the RhoA activation leads to the phosphorylation of Ocln, Cldn5 and TJ reorganization.⁴¹ It has been reported that the Cldn5 can be phosphorylated after the exposure of brain ECs to ROS.⁴⁰ The inhibition of Rho activation prevented the ROS-induced cytoskeleton rearrangements and the disappearance of Ocln and Cldn5. It indicates that ROS activates Rho, which subsequently may affect the actin cytoskeleton via PI3 kinase and PKB. Cytoskeleton depolymerization causes the redistribution of TJ molecules. Hence, signaling molecules that control the organization of the actin cytoskeleton may indirectly be involved in the regulation of TJs.³⁹

Quercetin, a flavonoid, has been shown to have a beneficial role in neuroprotection through strong antioxidant activity.⁴² It increases GSH level in brain and maintains spontaneous behavior and cognitive performance.⁴³ Our earlier studies reveal that quercetin acts against PCB-induced neurotoxicity in cerebral cortex, cerebellum and hippocampus by decreasing oxidative stress via scavenging ROS and brought back the messenger RNA (mRNA) expression of dopamine receptors in male rats.^9
–11PCB-induced neurodegeneration has also been assessed by histological studies. Our earlier studies proved that the exposure to PCBs decreased serum thyroid hormone profile, follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone and estradiol in male rats.⁴⁴ The present study is aimed to analyze the role of PCB on BBB integrity particularly on female rats. The studies on serum hormonal profiles and other parameters are in progress.

Materials and methods

Experimental design

Experiments were carried out on adult female Wistar rats weighing 180–200 g, which were maintained under standard laboratory conditions in large polypropylene animal cage. Six rats were kept in a cage and all animal had free access to pellet diet (Gold Mohur Ltd., Mumbai, India) ad libitum. Rats were maintained at room temperature (25°C) with normal light cycle (12 h light and 12 h dark) and treated for 30 days.

The animals were divided into the following groups: (1) group I comprises control rats that were injected with corn oil of 80 µl/day intraperitoneally (i.p.) for 30 days as a vehicle; (2) group II comprises rats that received i.p. injection of Aroclor 1254 at a dose of 2 mg/kg body weight (bwt)/day;¹³ group III comprises rats that were administered with Aroclor 1254 at a dose of 2 mg/kg bwt/day (i.p.) and simultaneously quercetin was supplemented gavage at a dose of 50 mg/kg bwt¹¹ and group IV comprises rats that received quercetin by gavage at a dose of 50 mg/kg bwt.¹¹ The doses of both Aroclor 1254 and quercetin were chosen based upon their activity in the in vivo functional and biochemical studies.^11,13 All animal procedures were approved by our institutional ethical committee (Ref No. IAEC No 01/01/11). After the treatment period of 30 days, rats were killed. The brain was dissected and cerebellum, cerebrum and hippocampus were isolated and analyzed for the mRNA expression of TJPs.

Drugs and chemicals

Aroclor 1254 was purchased from Chem Service (West Chester, Pennsylvania, USA). Quercetin, total RNA isolation (TRI) reagent and primers were purchased from Sigma-Aldrich Private Limited (Bangalore, Karnataka, India). Superscript-III reverse transcriptase was purchased from Invitrogen (Chennai, Tamilnadu, India) and PCR Ready Mix DNA Polymerase was purchased from KAPA-Biosystem (Chennai, Tamilnadu, India).

Oxidative stress markers

Estimation of H₂O₂

The generation of hydrogen peroxide (H₂O₂) was assayed by the method of Pick and Keisari.⁴⁵ The reaction mixture prepared consists of tissue homogenate, 2.64 ml phosphate buffer, 54 µl horseradish peroxidase, 30 µl of 28 nM phenol red, 165 µl of 5.5 nM dextrose and 600 µl of enzyme source. It was incubated at 35°C for 30 min and terminated by adding 60 µl of 10 N NaOH solution. Horseradish peroxidase converts H₂O₂ into water and oxygen. This causes the oxidation of phenol red, which forms adduct with dextrose, and shows maximum absorbance at 610 nm. The levels of H₂O₂ generation were expressed as nanomoles of H₂O₂ generated per minute per milligram of protein.

Estimation of LPO

Lipid peroxidation (LPO) was measured by the method of Devasagayam and Tarachand.⁴⁶ The reaction mixture comprising 0.2 ml of tissue homogenate, 1 ml of 0.15 M Tris-HCl buffer (pH 7.4), 0.3 ml of 10 mM potassium dihydrogen phosphate (KH₂PO₄) and 1.5 ml of 1% thiobarbituric acid (TBA) were heated for 20 min. The reaction was then stopped by the addition of 10% trichloroacetic acid. Malondialdehyde, an end product of LPO, reacts with TBA to form a pink chromogen (TBA 2-malondialdehyde adduct), and it was measured at 532 nm absorbance with a spectrophotometer. The results were expressed as nanomoles of malondialdehyde (MDA) formed per milligram of protein.

Measurement of TBARS as a marker of LPO

Cerebrum, cerebellum and hippocampus were excised out of the brain and rinsed with cold 0.14 M NaCl, and a part of it was homogenized in 25% ice-cold 50 mM Tris-HCl buffer, pH 7.4.⁴⁷ A total of 150 μL of the tissue supernatant of samples was diluted to 500 μL with deionized water. A total of 250 μL of 1.34% TBA was added to all the tubes, followed by the addition of an equal volume of 40% trichloroacetic acid. The mixture was shaken and incubated for 30 min in a boiling water bath. Tubes were allowed to cool to room temperature and the absorbance was read at 532 nm using zero concentration as blank. The results were expresses as nanomoles per milligram of protein. ⁴⁸

TRI and RT-PCR

Total RNA was isolated from cerebrum, cerebellum and hippocampus using TRI reagent following the method of Chomczynski and Sacchi.⁴⁹ Total RNA of 1 µg was subjected to two-step reverse transcriptase-polymerase chain reaction (RT-PCR). (1) First strand reaction: Complementary DNA (cDNA) is made from mRNA template using dNTPs and reverse transcriptase (Superscript-III Reverse Transcriptase, Invitrogen). The components were combined with a DNA primer in a reverse transcriptase buffer for an hour at 37°C. Second strand reaction: After the reverse transcriptase reaction was completed, cDNA has been generated from the original single strand mRNA, and the standard PCR was initiated (PCR Ready Mix DNA polymerase was purchased from KAPA-Biosystem, USA). PCR primers and conditions used in this study have been tabulated in Table 1. The amplified products were separated by electrophoresis on 2% agarose gel and identified by ethidium bromide staining. Specificity was confirmed by the size of the amplified products with reference to 100 bp DNA ladder (Chromus Biotech, Chennai, TN, India), and the band intensities were quantified by Quantity One Software (Bio-Rad, Gurgaon, Haryana, India).

Table 1.

List of primers

Target gene	Primer sequence (5′→3′)	Amplicon size (bp)	Annealing temperature (°C)	Gene bank /accession number
ZO-1	Sense: GGCTACAGCCCGCGGCATTT	213	56.7	NM_001106266.1
ZO-1	Antisense: CCATGGGGCCCGCACATCAC	213	56.7	NM_001106266.1
ZO-2	Sense: TCCCGGGACTACAGCCGTGG	208	59.1	NM_053773.1
ZO-2	Antisense: GCCGGGACCTAGACTCGGGG	208	59.1	NM_053773.1
Cldn5	Sense: CGTGACGGCGCAGACGACTT	194	57.8	NM_031701.2
Cldn5	Antisense: TGCACTGAGCGCCGGTCAAG	194	57.8	NM_031701.2
Ocln	Sense: TGGCGGAGAGATGCACGTTCG	247	55.2	NM_031329.2
Ocln	Antisense: ACCGAAGCCGCTGCCGTAAG	247	55.2	NM_031329.2
JAM-3	Sense: TCAAGGAGACCTGGCCGGTCG	254	62.9	NM_001004269.1
JAM-3	Antisense: GCCGAGGATAGCCCTCGCTCT	254	62.9	NM_001004269.1
AF6	Sense: GCGATGGGCGAGGCTGAAACA	180	62.9	NM_001007754.1
AF6	Antisense: ATCAGGTGTCTCCCGCTCCACAG	180	62.9	NM_001007754.1
β-actin	Sense: TCCACCCGCGAGTACAACCTTC	358	63.9	NM_031144.2
β-actin	Antisense : GGGCCACACGCAGCTCATTGTA	358	63.9	NM_031144.2

ZO-1/-2: zona occludens-1/-2; Ocln: occludin; Cldn5: claudin-5; JAMs: junction adhesion molecules; AF6: ALL-1 fusion partner at chromosome-6.

Statistical analysis

All the values were expressed as mean ± SEM of three independent observations. Data were analyzed using one-way analysis of variance followed by post hoc test Student’s Newman-Keul’s test with Graphpad Prism5 software. In all the cases, p < 0.05 was considered statistically significant.

Results

Effect of quercetin on body weight of adult female rats exposed to PCBs

The effect of PCBs on change in body weight of rats is shown in Figure 1. No mortality was observed in any of the experimental groups. Maximum weight gain was observed in the quercetin-administered rats when compared with all other groups. There was a gradual and significant (p < 0.05) decrease in the body weight of PCBs-treated rats after 20 days when compared with control and quercetin-treated rats.

Figure 1.

Effect of quercetin on the body weight of adult female rats exposed to PCBs. Each bar represents mean ± SEM of six animals. Statistical significance at p < 0.05. a: Control at 0 day vs. 10th, 20th and 30th day; b: Control at 10th day vs. 20th and 30th day. a′: PCB at 0 day vs. 10th, 20th and 30th day; b′: quercetin at 10th day vs. 20th and 30th day. a″: PCB + quercetin at 0 day vs. 10th, 20th and 30th day; b″: PCB at 10th day vs. 20th and 30th day. a′″: quercetin at 0 day vs. 10th, 20th and 30th day; b′″: PCB + quercetin at 10th day vs. 20th and 30th day; c′″: PCB + quercetin at 20th day vs. 30th day. PCB: polychlorinated biphenyls.

Effect of quercetin on oxidative stress markers in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs

The levels of H₂O₂ generation, LPO and TBA reactive substance (TBARS) content in cerebrum, cerebellum and hippocampal tissue of control and experimental rats were estimated (Figure 2(a) to (c)). The levels of H₂O₂, LPO and TBARS were increased significantly in PCB-exposed rats in cerebrum, cerebellum and hippocampus as 50%, 25% and 20%, respectively. Quercetin acts as a free radical scavenger and decreased the levels of oxidative stress markers (H₂O₂, LPO and TBARS) in quercetin alone or in PCB + quercetin-treated animals.

Figure 2.

Effect of quercetin on oxidative stress markers in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs. Each bar represents mean ± SEM of six animals. Statistical significance at p < 0.05. a: Control vs. others; b: PCB vs. quercetin, PCB+quercetin PCB: polychlorinated biphenyl.

mRNA expression of TJPs

Figure 3 represents the effect of quercetin on mRNA expression of integral membrane protein Ocln in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs. mRNA expression of Ocln was decreased (40%) in the exposure to Ocln, while simultaneous supplementation of quercetin brought the levels back to normal. Exposure to quercetin alone showed no change.

Figure 3.

Effect of quercetin on mRNA expression of integral membrane protein Ocln in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs. Each bar represents mean ± SEM of three independent observations. Statistical significance at p < 0.05. a: Control vs. others; b: PCB vs. others. M: 100 bp marker; L1: control; L2: PCB; L3: PCB + quercetin; L4: quercetin; PCB: polychlorinated biphenyl; Ocln: occludin; mRNA: messenger RNA.

Figure 4 represents the effect of quercetin on mRNA expression of integral membrane protein Cldn5 in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs. Cldn expression levels were decreased (40%) upon exposure to PCBs. Simultaneous supplementation of quercetin scavenges the ROS and decreases the degradation of Cldn and brought back to normal. Exposure to quercetin alone showed no change.

Figure 4.

Effect of quercetin on mRNA expression of integral membrane protein Cldn5 in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs. Each bar represents mean ± SEM of three independent observations. Statistical significance at p < 0.05. a: Control vs. others; b: PCB vs. others. M: 100 bp marker; L1: control; L2: PCB; L3: PCB + quercetin; L4: quercetin. PCB: polychlorinated biphenyl; Cldn5: claudin-5; mRNA: messenger RNA.

Figure 5 reveals the effect of quercetin on mRNA expression of integral membrane protein, that is, JAM-3 in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs. JAM levels were decreased upon exposure to PCBs, while simultaneous supplementation of quercetin brought the levels back to normal. A significant decrease (50%) was observed in cerebellum than in cerebrum (10%) and hippocampus (15%) of the rats exposed to PCBs. Quercetin alone did not show any significant change.

Figure 5.

Effect of quercetin on mRNA expression of integral membrane protein JAMs in cerebrum, cerebellum and hippocampus of PCBs exposed adult female rats. Each bar represents mean ± SEM of three independent observations. Statistical significance at p < 0.05. a: Control vs. others; b: PCB vs. others. M: 100 bp marker; L1: control; L2: PCB; L3: PCB + quercetin; L4: quercetin. PCB: polychlorinated biphenyl; JAMs: junction adhesion molecules; mRNA: messenger RNA.

Figure 6 represents the effect of quercetin on mRNA expression of cytoplasmic accessory proteins; zona occludens (ZO; ZO-1 and ZO-2) in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs. mRNA expression levels of both ZO-1 and ZO-2 were decreased upon exposure to PCBs, while simultaneous supplementation of quercetin brought the levels back to normal. Quercetin alone did not show any significant change. Similar to JAM, cerebellum showed 50% decrease in both ZO-1 and ZO-2 mRNA expressions after exposure to PCB than other regions.

Figure 6.

Effect of quercetin on mRNA expression of cytoplasmic accessory proteins: ZOs (ZO-1 and ZO-2) in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs. Each bar represents mean ± SEM of three independent observations. Statistical significance at p < 0.05. a: Control vs. others; b: PCB vs. others. M: 100 bp marker; L1: control; L2: PCB; L3: PCB + quercetin; L4: quercetin. PCB: polychlorinated biphenyl; mRNA: messenger RNA; ZOs: zona occludens.

Figure 7 shows the effect of quercetin on mRNA expression of cytoplasmic accessory proteins, such as AF6/afadin, in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs. mRNA expression levels of AF6 were decreased significantly in PCB-exposed rats in cerebrum, cerebellum and hippocampus as 30%, 50% and 15%, respectively. Quercetin supplementation is brought back only in cerebrum and hippocampus. In cerebellum, quercetin supplementation tried to become normal levels. Quercetin alone treated animals showed normal mRNA expression.

Figure 7.

Effect of quercetin on mRNA expression of cytoplasmic accessory protein (AF6) in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs. AF6: ALL-1 fusion partner at chromosome-6. Each bar represents mean ± SEM of three independent observations. Statistical significance at p < 0.05. a: Control vs. others; b: PCB vs. others. M: 100 bp marker; L1: control; L2: PCB; L3: PCB + quercetin; L4: quercetin. PCB: polychlorinated biphenyl; mRNA: messenger RNA.

Discussion

PCBs are the persistent organic pollutants that exhibited various toxic effects in animals and exposed human populations. The molecular mechanisms of PCB toxicity have been attributed to the toxicological properties of its metabolites, such as hydroquinone, formed by cytochrome-P-450 oxidation.⁵⁰ PCBs cause neurodegeneration via the production of ROS. The brain is especially vulnerable to oxidative damage for several reasons. The activities of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) are low in all the brain regions. Furthermore, the nerve cells are rich in polyunsaturated fatty acids that are especially sensitive to the attack by free radicals, which may lead to LPO and membrane impairment and which may lead to neurodenegerative diseases.¹³ PCBs cause neurodegeneration via the production of ROS.⁵¹ Since brain has low catalase, SOD and GPx activities, its membrane lipids are rich in polyunsaturated fatty acids, which are especially sensitive to free radical-induced LPO.⁵² Hence, the brain is more vulnerable to PCBs-induced neurodegeneration via ROS. This study prevails that H₂O₂, LPO and TBARS levels were significantly high after exposure to PCB. There are evidences suggesting that the oxidative stress induced by PCBs is due to the interaction of these compounds with AhR and activation of the cytochrome P450 1A superfamily.⁵³ Previous studies in our laboratory also suggest that PCBs–quinones undergo redox cycling with the formation of ROS, thus becoming a major source of oxidative stress in male Wistar rats. Earlier studies in our laboratory demonstrated that the exposure to PCB decreased mRNA expression of Cu/Zn, SOD and GPx-4 as well as histology of brain regions.⁵¹ PCB-induced oxidative stress decreases the activities of antioxidant enzymes and disrupts the functional parameters in ventral prostate, testicular Leydig cells, sertoli cells and selected brain regions,^{10,11,54

–58} and it can damage the cellular elements.

ROS may propagate the initial attack of lipid membranes of the brain to cause LPO. It is one of the manifestations of oxidative damage and it has been found to play an important role in the toxicity of several xenobiotics.⁵⁹ In the present study, in female rats, PCB increases ROS level by increased LPO, H₂O₂ and TBARS concentrations in cerebrum, cerebellum and hippocampus. Oxidative modification of proteins in vivo may affect a variety of cellular functions involving proteins: receptors, signal transduction mechanisms, transport systems and enzymes. It could also contribute to secondary damage to other biomolecules.

The BBB is anatomically situated at the level of the cerebral microvascular capillary endothelium and it regulates the blood–brain exchange. The BBB is primarily formed by specialized brain ECs, which form a tight seal due to the presence of well-developed TJs that impede the entrance of circulating molecules and immune cells into the CNS.⁶⁰ The TJs limit passive paracellular movement of solutes, ions and water across the BBB. This barrier is variable and physiologically regulated, and its disruption contributes to human diseases.⁶¹ There are ample evidences that PCBs cause neurotoxicity in humans. The predominant mechanisms include alteration in TJ of human brain microvascular ECs, intracellular-signaling process and thyroid hormone metabolism.⁶² The loss of TJ proteins is commonly observed in neuroinflammatory and neurodegenerative disorders that are frequently associated with stroke,⁶³ Alzheimer’s disease,⁶⁴ HIV-1 encephalitis⁶⁵ and traumatic brain injury.⁶⁶ Disruption of TJ in brain ECs can increase serum protein extravasation through impaired BBB and lead to the development of vasogenic brain edema. PCBs exposure induces disruption of TJ in human brain microvascular ECs, which may lead to perturbation of the BBB integrity.⁶⁷

The brain is particularly sensitive to oxidative damage because of its high levels of unsaturated lipids as well as a high rate of oxidative metabolism. ROS (oxy radicals) are generated continuously in nervous system during normal metabolism and neuronal activity.⁶⁸ Oxidative stress is a contributing factor for the alteration caused in neurodegenerative processes.⁵¹ PCB-induced toxic manifestations may be associated with the production of free radicals. Schilderman et al.⁶⁹ demonstrated that PCBs induce ROS which can damage the cellular elements in its target organs such as liver, spleen, seminal vesicles, prostate and epididymis. The ROS alter BBB integrity, which is paralleled by cytoskeleton rearrangements, redistribution and disappearance of TJ proteins³⁹ (ROS), which are highly reactive molecules, are produced during monocyte migration and contribute to BBB injury and subsequent inflammation in the brain.⁷⁰ In the present study, there is a decreased expression of TJ protein in the microvascular ECs in the cerebrum of PCB-treated rats.

TJs of the BBB composed of an intricate combination of integral membrane proteins (Ocln, Cldns and JAMs) mediate cellular interaction between brain ECs and play a major role in TJ functioning and cytoplasmic accessory proteins (ZO (ZO-1 and ZO-2) and AF6), providing a link between transmembrane TJ proteins and the actin cytoskeleton by participating in intracellular signaling. This study prevails that the mRNA expression of TJPs in cerebellum, cerebrum and hippocampus significantly decreased upon exposure to PCB. The evidence suggests that the PCB involves in the alteration of TJ of human brain microvascular ECs, intracellular-signaling process and thyroid hormone metabolism.⁶² The ROS alter BBB integrity, which is paralleled by cytoskeleton rearrangements, redistribution and disappearance of TJ proteins.³⁹ ROS, which are highly reactive molecules, are produced during monocyte migration and contribute to BBB injury and subsequent inflammation in the brain. PCBs-induced disruption of blood–brain homeostasis occurs predominantly by means of ROS-induced TJ disruption. The organization of TJ proteins Ocln and ZO-1 is altered by exogenous ROS in brain ECs.³⁸ ROS selectively activate signaling cascades involving RhoA, PI3 kinase and PKB/Akt leading to the rearrangements of actin cytoskeleton and spatial redistribution and disappearance of Ocln and Cldn5, inducing altered BBB integrity.³⁹ Previous studies show neuronal damage as well as alteration in neuronal morphology of cerebral cortical layer, pyramidal cells of hippocampal layers and the Purkinjee cellular layer in cerebellum after exposure to PCB.⁵⁶ It may be due to the enhanced free radical generation in PCB-exposed animals. Pyknotic nuclei with predominant perineuronal spaces were observed in cortical layer due to enhanced LPO. PCBs could also affect cerebellar development through complex actions on thyroid hormone system.⁷¹ Anbalagan et al.⁷² proved that the rats exposed to PCBs showed decreased thyroid hormone levels. In the present study, down regulation of both integral membrane proteins and cytoplasmic accessory proteins mRNA expression was observed in the PCBs-treated group. The possible mechanism behind this is the ROS-induced disruption.

Khan and Thomas⁷³ showed that the PCB inhibits tryptophan hydroxylase (TPH) activity, which is accompanied by the reduced 5 hydroxy tryptophan (5HT) concentrations in rat brain regions. PCB decreases TPH activity by a reduction in the enzyme protein inhibition of its biosynthesis or enhanced degradation to inhibit enzyme activation. They suggested that PCBs neurotoxicity may involve oxidative damage to TPH protein resulting in the reduction of TPH activity and 5HT concentration. In this present study, decreased junction proteins may be due to the inactivation of TPH protein by increasing LPO generation.

Quercetin is believed to work via electron donation to directly detoxify free radicals such as the highly toxic hydroxyl radicals. Quercetin is a strong oxygen radical scavenger and also a good metal chelator. In vitro studies have suggested that the quercetin has a potent inhibitory activity against the production of nitric oxide and tumor necrosis factor in lipopolysaccharide-stimulated Kupffer cells.⁷⁴ Quercetin was shown to scavenge superoxide in ischemia reperfusion injury.⁷⁵ Quercetin prevents oxidative stress-mediated diseases and inflammatory disorders. The 4-oxo group and 2,3-double bond in the c-ring of quercetin are thought to play an important role in their neuroprotective effects in repeated cerebral ischemic model.⁴³

Thus, toxicity of PCBs impairs TJP at the level of transcription by means of PKB and PI-3 kinase-signaling pathway related to ROS production. This PCB-induced neurotoxicity is minimized by quercetin, an antioxidant flavonoid. The antioxidant activity of flavonoids has been demonstrated by their abilities to scavenge free radicals⁷⁶ by inhibiting enzymes such as lipoxygenase and cycloxygenase and by chelating metal ions.⁷⁷ Selvakumar et al.⁵⁸ demonstrated that quercetin treatment can attenuate PCB-induced oxidative stress in the hippocampus of experimental animals. Quercetin exerts important role in providing indirect protection against free radical injury by stimulating antioxidant enzymes. The degeneration of cells was reduced in simultaneous administration of quercetin with PCB-exposed rats. In the present study, quercetin seems to be capable of protecting PCB-induced neurotoxic effects and maintains the TJP at the metabotrophic level.

Our earlier studies have proved that the PCBs induce ROS that can damage the cellular elements in its target organs such as brain, liver, spleen, kidney, seminal vesicles, prostate, epididymis, sperm, testicular sertoli and Leydig cells.^54

–59 Decreased antioxidant enzymes have also been studied in all the regions of brain in PCB-treated animals, indicating an increase in oxidative stress. PCB-induced neuronal damage on male rats has also been studied. However, the present study is focused on female rats. The neuroprotective role of quercetin in male rats has also been studied recently.^9
–11 Further study on female rats is warranted to understand whether quercetin prevents neuronal damage in brain regions. The present study infers that the quercetin protects PCB-induced BBB integrity and oxidative stress.

In the present study, disruption of TJ integrity due to PCB induction can occur from the loss of protein expression and/or disorganization. Dietary quercetin may exert its actions via the alteration of protein kinase activities and sequential-signaling processes. This may enable quercetin to prevent the disintegration of TJ structure and to enhance the expression, localization and interaction of TJ proteins.

Oxidative damages affect the expression, localization and organization of TJ proteins, particularly, Ocln, ZO1 and Cldns.⁷⁸ Oxidative stress is able to trigger signaling pathways in TJs regulation involving mitogen activated protein kinases (MAPKs), especially extracellular signal regulated kinases 1/2 (ERK1/2), P38 and c-jun NH2-terminal kinase (JNK), Protein kinase C (PKC) and phosphodiesterase.⁷⁹ Kevil et al.⁸⁰ studied that oxidative stress-induced hyperpermeability is related to the phosphorylation of Ocln and ZO1 at the tyrosine residues, down regulation of Ocln and activation of MAPK signaling pathways. The action of quercetin has been linked to a number of enzymes involved in the proliferation and signal transduction pathways including PKC, tyrosine kinase, PI3 kinase, nuclear factor kappa B (NFkß) and MPK family. Chuenkitiyanon et al.⁷⁹ studied that the protective effects of quercetin might involve the altered MAPK activities, in particular the decrease in p38 MAP signaling on epithelial and endothelial barriers.

PCBs have epigenetic effects on a wide variety of cellular and physiological functions. PCBs reduce DNA methylation in hippocampal neurons.⁸¹ Further studies are needed. Study on the involvement of protein kinase C and MAPK-signaling pathway after quercetin supplementation is warranted.

Conclusion

PCBs disrupt the gene expression of transmembrane TJPs (Ocln, Cldn5 and JAM) and cytoplasmic accessory TJPs (ZO-1, ZO-2 and AF6) on cerebrum, cerebellum and hippocampus. Quercetin scavenges the PCBs-induced ROS, thereby preventing the transmembrane TJPs and cytoplasmic accessory TJPs in cerebrum, cerebellum and hippocampus from degradation. Protein expression of all these TJ proteins will prove the PCB disruption in a more authentic manner.

Footnotes

Conflict of Interest

The authors declared no conflicts of interest.

Funding

The financial assistance is obtained from the University Grants Commission-Research Fellowship in Science for meritorious students (UGC-RFSMS) program (to KS).

References

Seelbach

Chen

Powell

Choi

Zhang

Hennig

Polychlorinated biphenyls disrupt blood-brain barrier integrity and promote brain metastasis formation. Environ Health Perspect 2010; 118: 479–484.

Safe

. Toxicology, structure-function relationship, and human and environmental health impacts of polychlorinated biphenyls: progress and problems. Environ Health Perspect 1993; 100: 259–268.

Schneider

Porter

Baker

. Polychlorinated biphenyl release from resuspended Hudson river sediment. Environ Sci Technol 2007; 41: 1097–1103.

Webb

McCall

. Identities of polychlorinated biphenyl isomers in aroclors. J Assoc Off Anal Chem 1972; 55: 746–752.

Mariussen

Myhre

Reistad

Fonnum

. The polychlorinated biphenyl mixture aroclor 1254 induces death of rat cerebellar granule cells: the involvement of the N-methyl-D-aspartate receptor and reactive oxygen species. Toxicol Appl Pharmacol 2002; 179: 137–144.

Kaya

Hany

Fastabend

Roth-Harer

Winneke

Lilienthal

. Effects of maternal exposure to a reconstituted mixture of polychlorinated biphenyls on sex-dependent behaviors and steroid hormone concentrations in rats: dose-response relationship. Toxicol Appl Pharmacol 2002; 178: 71–81.

Stewart

Reihman

Lonky

Darvill

Pagano

. Prenatal PCB exposure and neonatal behavioral assessment scale (NBAS) performance. Neurotoxicol Teratol 2000; 22: 21–29.

Kodavanti

Tilson

. Neurochemical effects of environmental chemicals: in vitro and in vivo correlations on second messenger pathways. Ann N Y Acad Sci 2000; 919: 97–105.

Pratheepa Kumari

Selvakumar

Bavithra

Zumaana

Krishnamoorthy

Arunakaran

. Role of quercetin on PCBs (Aroclor-1254) induced impairment of dopaminergic receptor mRNA expression in cerebral cortex of adult male rats. Neurochem Res 2011; 36: 1344–1352.

10.

Bavithra

Selvakumar

Pratheepa Kumari

Krishnamoorthy

Venkataraman

Arunakaran

. Polychlorinated biphenyl (PCBs)-induced oxidative stress plays a critical role on cerebellar dopaminergic receptor expression: ameliorative role of quercetin. Neurotox Res 2012; 21: 149–159.

11.

Selvakumar

Bavithra

Krishnamoorthy

Venkataraman

Arunakaran

. Polychlorinated biphenyls-induced oxidative stress on rat hippocampus: a neuroprotective role of quercetin. Scientific World J 2012a; 2012: 980314.

12.

Sridevi

Venkataraman

Senthilkumar

Krishnamoorthy

Arunakaran

. Oxidative stress modulates membrane bound ATPases in brain regions of PCB (Aroclor 1254) exposed rats: protective role of alpha-tocopherol. Biomed Pharmacother 2007; 61: 435–440.

13.

Venkataraman

Krishnamoorthy

Vengatesh

Srinivasan

Aruldhas

Arunakaran

. Protective role of melatonin on PCB (Aroclor 1,254) induced oxidative stress and changes in acetylcholine esterase and membrane bound ATPases in cerebellum, cerebral cortex and hippocampus of adult rat brain. Int J Dev Neurosci 2008; 26: 585–591.

14.

Abbott

. Dynamics of CNS barriers: evolution, differentiation, and modulation. Cell Mol Neurobiol 2005; 25: 5–23.

15.

Pardridge

. Drug and gene delivery to the brain: the vascular route. Neuron 2002; 36: 555–558.

16.

Stevenson

Siliciano

Mooseker

Goodenough

. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 1986; 103: 755–766.

17.

Denker

Nigam

. Molecular structure and assembly of the tight junction. Am J Physiol 1998; 274: F1–F9.

18.

Wolburg

Lippoldt

. Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol 2002; 38: 323–337.

19.

Chen

Pothoulakis

LaMont

. Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A. J Biol Chem 2002; 277: 4247–4254.

20.

Liu

LeCluyse

Thakker

. Dodecylphosphocholine-mediated enhancement of paracellular permeability and cytotoxicity in Caco-2 cell monolayers. J Pharm Sci 1999; 88: 1161–1168.

21.

Hollande

Blanc

Bali

Whitehead

Pelegrin

Baldwin

HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line. Am J Physiol Gastrointest Liver Physiol 2001; 280: G910–G921.

22.

Blum

Toninelli

Anderson

Balda

Zhou

O'Donnell

Cytoskeletal rearrangement mediates human microvascular endothelial tight junction modulation by cytokines. Am J Physiol 1997; 273: H286–H294.

23.

Fischer

Wiesnet

Marti

Renz

Schaper

. Simultaneous activation of several second messengers in hypoxia-induced hyperpermeability of brain derived endothelial cells. J Cell Physiol 2004; 198: 359–369.

24.

Balda

Matter

. The tight junction protein ZO-1 and an interacting transcription factor regulate ErbB-2 expression. EMBO J 2000; 19: 2024–2033.

25.

Ryeom

Paul

Goodenough

. Truncation mutants of the tight junction protein ZO-1 disrupt corneal epithelial cell morphology. Mol Biol Cell 2000; 11: 1687–1696.

26.

Hawkins

Davis

. The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 2005; 57: 173–185.

27.

Betanzos

Huerta

Lopez-Bayghen

Azuara

Amerena

Gonzalez-Mariscal

The tight junction protein ZO-2 associates with Jun, Fos and C/EBP transcription factors in epithelial cells. Exp Cell Res 2004; 292: 51–66.

28.

Hom

Fleegal

Egleton

Campos

Hawkins

Davis

. Comparative changes in the blood-brain barrier and cerebral infarction of SHR and WKY rats. Am J Physiol Regul Integr Comp Physiol 2007; 292: R1881–R1892.

29.

Kanmogne

Primeaux

Grammas

. HIV-1 gp120 proteins alter tight junction protein expression and brain endothelial cell permeability: implications for the pathogenesis of HIV-associated dementia. J Neuropathol Exp Neurol 2005; 64: 498–505.

30.

Andras

Deli

Nath

Hennig

Toborek

. HIV-1 Tat protein alters tight junction protein expression and distribution in cultured brain endothelial cells. J Neurosci Res 2003; 74: 255–265.

31.

Boettner

Govek

Cross

Van Aelst

. The junctional multidomain protein AF-6 is a binding partner of the Rap1A GTPase and associates with the actin cytoskeletal regulator profilin. Proc Natl Acad Sci USA 2000; 97: 9064–9069.

32.

Yamamoto

Harada

Kano

Taya

Canaani

Matsuura

The ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells. J Cell Biol 1997; 139: 785–795.

33.

Zhang

Rehmann

Price

Riedl

Bos

. AF6 negatively regulates Rap1-induced cell adhesion. J Biol Chem 2005; 280: 33200–33205.

34.

Carpenter

. Polychlorinated biphenyls (PCBs): routes of exposure and effects on human health. Rev Environ Health 2006; 21: 1–23.

35.

Knerr

Schrenk

. Carcinogenicity of “non-dioxinlike” polychlorinated biphenyls. Crit Rev Toxicol 2006; 36: 663–694.

36.

Lee

Opanashuk

. Polychlorinated biphenyl mixture aroclor 1254-induced oxidative stress plays a role in dopaminergic cell injury. Neurotoxicology 2004; 25: 925–939.

37.

Van der Goes

Wouters

Van Der Pol

Huizinga

Ronken

Adamson

Reactive oxygen species enhance the migration of monocytes across the blood-brain barrier in vitro. FASEB J 2001; 15: 1852–1854.

38.

Fischer

Wiesnet

Renz

Schaper

. H₂O₂ induces paracellular permeability of porcine brain-derived microvascular endothelial cells by activation of the p44/42 MAP kinase pathway. Eur J Cell Biol 2005; 84: 687–697.

39.

Schreibelt

Kooij

Reijerkerk

van Doorn

Gringhuis

van der Pol

Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling. FASEB J 2007; 21: 3666–3676.

40.

Haorah

Ramirez

Schall

Smith

Pandya

Persidsky

. Oxidative stress activates protein tyrosine kinase and matrix metalloproteinases leading to blood-brain barrier dysfunction. J Neurochem 2007; 101: 566–576.

41.

Jou

Schneeberger

Nelson

. Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases. J Cell Biol 1998; 142: 101–115.

42.

Cho

Kim

Jang

Kim

Lee

. Protective effect of quercetin, a natural flavonoid against neuronal damage after transient global cerebral ischemia. Neurosci Lett 2006; 404(3): 330–335.

43.

Mishima

Irie

Motohashi

Tanaka

Orito

Neuroprotective effects of quercetin and rutin on spatial memory impairment in an 8-arm radial maze task and neuronal death induced by repeated cerebral ischemia in rats. J Pharmacol Sci 2007; 104: 329–334

44.

Venkataraman

Sridhar

Dhanammal

Vijayababu

Arunkumar

Srinivasan

Effects of vitamin supplementation on PCB (Aroclor 1254)-induced changes in ventral prostatic androgen and estrogen receptors. Endocr Res 2004; 3: 469–480.

45.

Pick

Keisari

Superoxide anion and H2O2 production by chemically elicited peritoneal macrophages-induction by multiple nonphagocytic stimuli. Cell Immunol 1981; 59: 301–318

46.

Devasagayam

Tarachand

. Decreased lipid peroxidation in rat kidneys during gestation. Biochem Biophy Res Comm 1987; 145: 134–138.

47.

Benjamin

Iwata

Hazalett

. Kinetics of entry of proteins into the myelin membrane. J Neurochem 1978; 31: 1077–1085.

48.

Gutteridge

Quinlan

. Malondialdhyde formation from lipid peroxides in the thiobarbituric acid test: the role of lipid radicals, iron salt and metal chelators. J Appl Biochem 1983; 5 (4–5): 293–299.

49.

Chomczynski

Sacchi

. The single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction: twenty-something years on. Nat Protoc 2006; 1: 581–585.

50.

Chan

Lehmler

Sivagnanam

Feng

Robertson

Cytotoxic effects of polychlorinated biphenyl hydroquinone metabolites in rat hepatocytes. J Appl Toxicol 2010; 30: 163–171.

51.

Venkataraman

Muthuvel

Krishnamoorthy

Arunkumar

Sridhar

Srinivasan

PCB (Aroclor 1254) enhances oxidative damage in rat brain regions: protective role of ascorbic acid. Neurotoxicology 2007; 28: 490–498.

52.

Floyd

Leblanc

Whittaker

. Combinatorial chemistry as a tool for drug discovery. Prog Med Chem 1999; 36: 91–168.

53.

Alsharif

Lawson

Stohs

. Oxidative stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin is mediated by the aryl hydrocarbon (Ah) receptor complex. Toxicology 1994; 92: 39–51.

54.

Sridhar

Venkataraman

Dhanammal

Arunkumar

Aruldhas

Srinivasan

Impact of polychlorinated biphenyl (Aroclor 1254) and vitamin C on antioxidant system of rat ventral prostate. Asian J Androl 2004; 6: 19–22.

55.

Murugesan

Senthilkumar

Balasubramanian

Aruldhas

Arunakaran

. Impact of polychlorinated biphenyl Aroclor 1254 on testicular antioxidant system in adult rats. Hum Exp Toxicol 2005; 24: 61–66.

56.

Venkataraman

Selvakumar

Krishnamoorthy

Muthusami

Rameshkumar

Prakash

Effect of melatonin on PCB (Aroclor 1254) induced neuronal damage and changes in Cu/Zn superoxide dismutase and glutathione peroxidase-4 mRNA expression in cerebral cortex, cerebellum and hippocampus of adult rats. Neurosci Res 2010; 66: 189–197.

57.

Selvakumar

Sheerin Banu

Krishnamoorthy

Venkataraman

Elumalai

Arunakaran

. Differential expression of androgen and estrogen receptors in PCB (Aroclor 1254)-exposed rat ventral prostate: impact of alpha-tocopherol. Exp Toxicol Pathol 2011; 63: 105–112.

58.

Selvakumar

Bavithra

Suganthi

Benson

Elumalai

Arunkumar

Protective role of quercetin on PCBs-induced oxidative stress and apoptosis in hippocampus of adult rats. Neurochem Res 2012b; 37: 708–721.

59.

Anane

Creppy

. Lipid peroxidation as pathway of aluminium cytotoxicity in human skin fibroblast cultures: prevention by superoxide dismutase? Catalase and vitamins E and C. Hum Exp Toxicol 2001; 20: 477–481.

60.

Pachter

de Vries

Fabry

. The blood-brain barrier and its role in immune privilege in the central nervous system. J Neuropathol Exp Neurol 2003; 62: 593–604.

61.

Powell

. Barrier function of epithelia. Am J Physiol 1981; 241: G275–G288.

62.

Kodavanti

. Neurotoxicity of persistent organic pollutants: possible mode(s) of action and further considerations. Dose Response 2005; 3: 273–305.

63.

Brown

Davis

. Calcium modulation of adherens and tight junction function: a potential mechanism for blood-brain barrier disruption after stroke. Stroke 2002; 33: 1706–1711.

64.

Fiala

Liu

Sayre

Pop

Brahmandam

Graves

Cyclooxygenase-2-positive macrophages infiltrate the Alzheimer’s disease brain and damage the blood-brain barrier. Eur J Clin Invest 2002; 32: 360–371.

65.

Persidsky

Heilman

Haorah

Zelivyanskaya

Persidsky

Weber

Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE). Blood 2006; 107: 4770–4780.

66.

Morganti-Kossmann

Rancan

Stahel

Kossmann

. Inflammatory response in acute traumatic brain injury: a double-edged sword. Curr Opin Crit Care 2002; 8: 101–105.

67.

Eum

Andras

Couraud

Hennig

Toborek

. PCBs and tight junction expression. Environ Toxicol Pharmacol 2008; 25: 234–240.

68.

Allen

Tresini

. Oxidative stress and gene regulation. Free Radic Biol Med 2000; 28: 463–499.

69.

Schilderman

Maas

Pachen

De Kok

Kleinjans

Van Schooten

. Induction of DNA adducts by several polychlorinated biphenyls. Environ Mol Mutagen 2000; 36: 79–86.

70.

Schreibelt

Musters

Reijerkerk

de Groot

van der Pol

Hendrikx

Lipoic acid affects cellular migration into the central nervous system and stabilizes blood-brain barrier integrity. J Immunol 2006; 177: 2630–2637.

71.

Brouwer

Morse

Lans

Schuur

Murk

Klasson-Wehler

Interactions of persistent environmental organohalogens with the thyroid hormone system: mechanisms and possible consequences for animal and human health. Toxicol Ind Health 1998; 14: 59–84.

72.

Anbalagan

Kanagaraj

Srinivasan

Aruldhas

Arunakaran

Effect of polychlorinated biphenyl, Aroclor 1254 on rat epididymis. Indian J Med Res 2003; 118: 236–242.

73.

Khan

Thomas

. Aroclor 1254 inhibits tryptophan hydroxylase activity in rat brain. Arch Toxicol 2004; 78: 316–320.

74.

Kawada

Seki

Inoue

Kuroki

. Effect of antioxidants, resveratrol, quercetin, and N-acetylcysteine, on the functions of cultured rat hepatic stellate cells and Kupffer cells. Hepatology 1998; 27: 1265–1274.

75.

Huk

Brovkovych

Nanobash Vili

Weigel

Neumayer

Partyka

Bioflavonoid quercetin scavenges superoxide and increases nitric oxide concentration in ischaemia-reperfusion injury: an experimental study. Br J Surg 1998; 85: 1080–1085.

76.

Guardia

Rotelli

Juarez

Pelzer

. Anti-inflammatory properties of plant flavonoids. Effects of rutin, quercetin and hesperidin on adjuvant arthritis in rat. Farmaco 2001; 56: 683–687.

77.

Yokozawa

, Rhyu DY and Cho EJ. (-)-Epicatechin 3-O-gallate ameliorates the damages related to peroxynitrite production by mechanisms distinct from those of other free radical inhibitors. J Pharm Pharmacol 2004; 56: 231–239.

78.

Krizbai

Bauer

Bresgen

Eckl

Farkas

Szatmári

Effect of oxidative stress on the junctional proteins of cultured cerebral endothelial cells. Cell Mol Neurobiol 2005; 25: 129–139.

79.

Chuenkitiyanon

Pengsuparp

Jianmongkol

. Protective effect of quercetin on hydrogen peroxide-induced tight junction disruption. Int J Toxicol 2010; 29: 418–424.

80.

Kevil

Oshima

Alexander

. The role of p38 MAP kinase in hydrogen peroxide mediated endothelial solute permeability. Endothelium 2001; 8: 107–116.

81.

Zhang

. Epigenetics meets endocrinology. J Mol Endocrinol 2011; 46: R11–R32.

Polychlorinated biphenyls impair blood–brain barrier integrity via disruption of tight junction proteins in cerebrum,cerebellum and hippocampus of female Wistar rats

Abstract

Keywords

Introduction

Materials and methods

Experimental design

Drugs and chemicals

Oxidative stress markers

Estimation of H2O2

Estimation of LPO

Measurement of TBARS as a marker of LPO

TRI and RT-PCR

Statistical analysis

Results

Effect of quercetin on body weight of adult female rats exposed to PCBs

Effect of quercetin on oxidative stress markers in cerebrum, cerebellum and hippocampus of adult female rats exposed to PCBs

mRNA expression of TJPs

Discussion

Conclusion

Footnotes

Conflict of Interest

Funding

References

Estimation of H₂O₂