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. Author manuscript; available in PMC: 2024 Jul 15.
Published in final edited form as: J Hazard Mater. 2023 Apr 26;454:131499. doi: 10.1016/j.jhazmat.2023.131499

Exposure to polychlorinated biphenyls selectively dysregulates endothelial circadian clock and endothelial toxicity

Timea Teglas 1, Silvia Torices 1, Madison Taylor 1, Desiree Coker 1, Michal Toborek 1,2
PMCID: PMC10202419  NIHMSID: NIHMS1899188  PMID: 37126901

Abstract

Polychlorinated biphenyls (PCBs) are lipophilic and persistent environmental toxicants, which pose health threats to the exposed population. Among several organs and cell types, vascular tissue and endothelial cells are especially prone to PCB-induced toxicity. Exposure to PCBs can exert detrimental impacts on biological pathways, expression of transcription factors, and tight junction proteins that are integral to the functionality of endothelial cells. Because biological and cellular processes are tightly regulated by circadian rhythms, and disruption of the circadian system may cause several diseases, we evaluated if exposure to PCBs can alter the expression of the major endothelial circadian regulators. In addition, we studied if dysregulation of circadian rhythms by silencing the brain and muscle ARNT-like 1 (Bmal1) gene can contribute to alterations of brain endothelial cells in response to PCB treatment. We demonstrated that diminished expression of Bmal1 enhances PCB-induced dysregulation of tight junction complexes, such as the expression of occludin, JAM-2, ZO-1, and ZO-2 especially at pathologically relevant longer PCB exposure times. Overall, the obtained results imply that dysregulation of the circadian clock is involved in endothelial toxicity of PCBs. The findings provide new insights for toxicological studies focused on the interactions between environmental pollutants and regulation of circadian rhythms.

Keywords: Polychlorinated biphenyls, environmental pollutants, tight junction, Bmal1, circadian rhythm

Graphical Abstract

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1. Introduction

Polychlorinated biphenyls (PCBs) have been widely used for industrial applications until the 1970s, when research shed light on their toxicity and resistance to biological decomposition (IARC, 2016). PCBs are highly toxic, lipophilic compounds, and their lipophilicity guides their accumulation in human and animal lipid tissues, as well as in the environment (Fitzgerald et al., 2007). PCBs are present on each level of the food chain, with the major source of PCB intake being the contaminated foods consumed, including all classes of animal products (Desvignes et al., 2015; Ghosh et al., 2014).

There are 209 recognized PCB congeners and their structural differences depend on the chemical position of the chlorine molecules (Carpenter, 2006). The position and degree of chlorine substitution(s) on two linked benzene rings group PCB congeners into three groups: the coplanar, the non-coplanar, and mono-ortho PCBs. The coplanar PCBs have chlorine substitutes in the para and meta positions, which determine their coplanar conformational character and flat structure. Environmentally, the most common coplanar PCB is 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) (Van den Berg et al., 2006). Mono-ortho-PCBs have a single ortho-chlorine substitution into the biphenyl ring, which disturbs coplanarity between the rings. A typical example is 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118). Biologically, mono-ortho-PCBs can interact with both coplanar and non-coplanar cellular receptors. The non-coplanar PCBs have more than one chlorine substitution in the ortho position. The most common non-coplanar environmental PCB is 2,2′,4,4′,5,5’-hexachlorobiphenyl (PCB 153) (Kraft et al., 2017). Structural variations of PCB congeners lead to differential interactions with biological targets and pathways, which then determine specific adverse health effects (Faroon and Ruiz, 2016). The coplanar PCBs bind to the Ah receptor (AhR) and induce the AhR-related signaling pathway (Carpenter, 2006). The non-coplanar PCBs are also called phenobarbital-like, ortho-substituted, or non-dioxin-like PCBs. They interact with the constitutive androstane receptor (CAR) and the pregnane-xenobiotic receptor (PXR) (Grimm et al., 2015; Klocke and Lein, 2020). PCBs have been consistently identified in human and animal blood and, in highest concentrations, in lipid tissue (Sontag et al., 2021). While PCBs are pervasive and remain in the environment for a long time (Fang et al., 2020; Gabryszewska and Gworek, 2020; Salvo et al., 2020), recent reports indicated that natural disasters, such as hurricanes or floods, can lead to redistribution of environmental PCBs from sediments and/or storage sites and increase human exposure (Cassin et al., 2018; Seidl et al., 2022; Wang et al., 2019). Several studies have identified associations of PCB exposure with human health effects such as neurological, cardiovascular and metabolic disorders (Dirinck et al., 2011; Sergeev and Carpenter, 2005; Simhadri et al., 2020; Wang et al., 2008).

Many physiological processes display day-night rhythms, including lipid/carbohydrate metabolism, feeding behavior, sleeping patterns, and hormone retention which processes are coordinated and followed 24-hour rhythm by the endogenous circadian clock. The molecular circadian oscillators are driven by negative feed-back loops that are largely controlled by the brain and muscle ARNT-like 1 (Bmal1) and the circadian locomotor output cycles kaput (Clock). These transcription factors form a heterodimer, which regulates the promoter activity of circadian-regulated genes. For example, the Bmal1/Clock complex affects the expression of Per (period) and Cry (cryptochrome), which then diminishes Bmal1/Clock activity via a feedback inhibition (Gekakis et al., 1998; Ko and Takahashi, 2006). Because of the importance of maintaining circadian system, several safeguards are in place and most circadian genes have paralogs, such as Per1 and Per2 or Cry1 and Cry2, meaning that both genes of the pair have to be silenced to achieve arrhythmicity. For example, a close paralog of Clock gene, the neuronal PAS domain protein 2 (Npas2) can compensate for lack of Clock gene expression, which is a functionally redundant paralog of the Clock gene (DeBruyne et al., 2007). A prominent exception to this pattern is Bmal1 and its paralog, Bmal2. Indeed, the loss of Bmal1 downregulates Bmal2 expression, abolishes clock function, and leads to arrhythmia (Shi et al., 2010).

The literature data on the impact of environmental toxicants on dysregulation of circadian clock involved in endothelial toxicity is very limited. Nevertheless, studies showed that the environmental contaminants, including hydroxylated metabolites of PCBs (Ochiai et al., 2018a), environmental progestins (Zhao et al., 2015), and metals (Hargreaves et al., 2022) can affect the circadian rhythms. The circadian rhythms can be dysregulated by environmental events and genetic factors, and these processes can contribute to the development of diseases, including cerebrovascular and neurological disorders (Crnko et al., 2019; Walker et al., 2020). Based on these results, this present study investigated the potential impact of PCB exposure on circadian rhythms in brain endothelial cells and determined how disruption of circadian rhythms can affect endothelial toxicity of PCBs. Our results indicate that treatment with PCBs disrupts expression patterns of circadian genes. Moreover, circadian disruption achieved by Bmal1 silencing potentiates PCB-induced alterations of endothelial integrity via dysregulation of tight junction complexes, especially the expression of occludin, zonula occludens (ZO)-1 and ZO-2.

2. Materials and methods

2.1. Brain endothelial cells

The human brain capillary endothelial cell line (hCMEC/D3 cells; a gift from Dr. Couraud; Institut Cochin, Paris, France) were cultured in Endothelial Cell Basal Medium-2 (EBM-2, #CC-3156 and EGMTM-2 Endothelial SingleQuots™ Kit #CC-4176, both from Lonza, Walkersville, MD, USA) in collagen-coated dishes (#C3867-1VL, Sigma-Aldrich, St. Louis, MO, USA) at 37°C with 5% CO2.

2.2. Polychlorinated biphenyl (PCB) treatment

3,3′,4,4′,5-Pentachlorobiphenyl (PCB126, #C-126S, #Lot051214KC), 2,2′,3,4,4′,5′-hexachlorobiphenyl (PCB138, #C-138N, #Lot121107MT-AC-01), 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153, #C-153N, #Lot110205KS-AC) and 2,2′,3,4,4′,5,5′-hexachlorobiphenyl (PCB180, #C-180N, #Lot021211KS-01) were purchased from AccuStandard (> 99.00% purity, New Haven, CT, USA) and dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA). PCBs were produced as mixtures, such as Aroclor 1260, which contained primarily noncoplanar PCB congeners, such as those used in the present study. Therefore, in order to reflect environmental exposure, cells were exposed not to a single PCB congener, but rather to a mix of PCB138, PCB153 and PCB180 in the molar ratio of 1.7:3.2:1 and the concentration of 5 μM. These are the most environmentally common PCBs and most frequently detected di-ortho-chlorine-substituted PCBs in population studies (CDC, 2009; Heudorf et al., 2002; Patterson et al., 1994; Turyk et al., 2006). In fact, they serve as, so called indicator congeners when measured in tissues (Bates et al., 2004; Glynn et al., 2007; Longnecker et al., 2000). In the US population, these three PCBs account for 65% of the measured total sum of PCBs (Needham et al., 2005) and for 78% of the total in a studied population of Italian residents in 2001-2003 (Apostoli et al., 2005). The employed concentration reflects serum PCB levels in acutely exposed human populations (Jensen, 1989; Wassermann et al., 1979), and brain endothelial cells are directly exposed to PCBs circulating in the blood-stream. Moreover, PCBs at 5 μM do not affect cell viability (Choi et al., 2013). Because of the importance of coplanar PCBs to the overall PCB toxicity, the mix of PCB138, PCB 153, and PCB180 was enriched with PCB126 at 0.1% as the additional treatment group. The dose of PCB 126 was chosen based on the serum PCB levels measured in the National Health and Nutrition Examination Survey (NHANES) 2003-2004 (Cave et al., 2010). The same amount of DMSO (vehicle, less than 0.1%) was added to control cultures.

2.3. Bmal1 silencing

Cells were subcultured and transferred into six-well plates at a density of 500,000 cells/cm2 per well when they reached 95% confluency. Cells were grown to 80% confluence for one day before transfection. Cultures were synchronized by staggering at different time points. The transfection mix was prepared in Opti-MEM (#11058021, Invitrogen, Carlsbad, CA, USA) with added Silencer Negative Control (#AM4611, Ambion, Austin, TX, USA) or Bmal1 siRNA (#L-012977-00-0005, Dharmacon, Lafayette, CO, USA), along with Lipofectamine RNAiMAX (#13778-075, Invitrogen, Carlsbad, CA, USA). The final concentration of siRNA and Lipofectamine added to the cells was 75 nM and 7.5 μL/mL, respectively. Cells were transfected for 6 hours, followed by recovery in EBM-2 complete medium for additional 18 hours. Cells were exposed to PCB mixtures 24 h after start of transfection (Figure S1).

2.4. Western Blotting

Treated cultures were lysed in RIPA buffer (#sc-24948, RIPA Buffer System, Santa Cruz Biotechnology, Dallas, TX, USA), incubated for 30 min on ice, followed by centrifugation at 15,000xg for 15 min and protein measurement in the supernatant by the BCA™ Assay Kit (Pierce, Rockford, IL, USA). For immunoblotting, proteins (10 μg) were separated on the 4-6% SDS-polyacrylamide gels and transferred onto 0.2 μM pore size polyvinylidene fluoride membranes (both from Bio-Rad, Hercules, CA, USA), which were blocked for 1 h with a blocking buffer consisting of 5% bovine serum albumin (BSA) in 1x TBS (VWR, Radnor, PA, USA) with 0.05% Tween 20 (TBST, Sigma-Aldrich, St. Louis, MO, USA). The samples were then incubated with primary antibodies (Table S1) overnight at 4°C. The membranes were washed with TBST and incubated for 1 h at 4°C with mouse or rabbit secondary antibodies (diluted at 1:20,000, #926-32210 IRDye 800CW Goat anti-Mouse, #926-68070 IRDye 680RD Goat anti-Mouse, #926-32211 IRDye 800CW Goat anti-Rabbit, #926-68071 IRDye 680RD Goat anti-Rabbit, all from LI-COR, Lincoln, NE, USA).

2.5. Immunofluorescence microscopy, confocal imaging, and imagine analysis

The cells were fixed for 15 min in 4% paraformaldehyde (Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature (RT). After extensive washing, they were permeabilized with 0.1% Triton-100 (Santa Cruz Biotechnology, Dallas, TX, USA) for 15 min and unspecific binding was blocked with 3% BSA in 1x PBS at RT (VWR, Radnor, PA, USA). Then, cells were incubated overnight (4 °C) with 1:100 β-catenin monoclonal antibody (#ab16051, Abcam, Woburn, MA, USA) in a humidified atmosphere. The following day, after 1x PBS washing, coverslips were incubated for 2 h with Alexa Fluor 647 (1:300, Thermo Fisher Scientific, Waltham, MA, USA) secondary antibodies at RT. Following extensive washing (1x PBS), the slides were mounted with Vectashield HardSet Antifade Mounting Medium with DAPI (Vector Labs, Burlingame, CA, USA). Confocal microscopy (FV3000, Olympus, Tokyo, Japan) was used to capture images and ImageJ (National Institutes of Health, Bethesda, MA, USA) was employed to analyze the images.

2.6. RT-qPCR

Total RNA was extracted using RNeasy Kit (Qiagen, Germany), followed by the reverse transcription reaction with the qScript XLT 1-Step RT-qPCR ToughMix Low ROX (Quantabio, Beverly, MA, USA). Table S2 lists the primers that were used for real-time PCR amplification. The expression of GAPDH was chosen as a house-keeping gene (VIC, Thermo Fisher Scientific, Waltham, MA, USA).

2.7. Statistics Analysis

The results were analyzed by one-way Analysis of Variance (ANOVA) using Prism software. p-values ≤ 0.05 were considered to be significant. Results were expressed as means ± Standard Error of Mean (SEM).

3. Results

3.1. Exposure to PCBs selectively upregulates circadian rhythm genes

We first characterized the expression levels of the core clock genes after PCB exposure. After synchronization, the cells were treated with vehicle or one of the following distinct PCB combinations: (i) PCB mix (a mix of non-coplanar PCBs) at 5 μM, (ii) PCB mix at 5 μM + PCB126 at 0.1%, or (3) PCB126 at 0.1%. PCBs at the concentration of 5 μM do not affect viability of brain endothelial cells (Eum et al., 2009). In addition, they are consistent with the serum levels in humans who were acutely exposed to these toxicants (CDC, 2009; Greig, 1991; Wassermann et al., 1979). mRNA expression of Clock, Bmal1, Cryl, Cry2, Perl, and Per2 were analyzed by RT-qPCR at 1h, 3h, 6h, 12h, 24h, 48h, and 72h after PCB treatment (Figures 1A–F, respectively). Expression of the circadian genes showed a characteristic waving pattern at different time points of PCB exposure. mRNA levels for Clock, Bmal1, Cry1 and Per2 were typically increased after 6h and/or 12 h of PCB treatment, then returned to control levels and typically slightly increased again after 48h of treatment. In general, the heterodimeric transcriptions factors, Clock and Bmal1, showed similar expression changes after PCB treatment. At 6 and/or 12h, the most pronounced elevation of circadian genes was observed in cells exposed to non-coplanar PCB mix, and the addition of PCB126 did not markedly influence these effects. However, the impact of PCB126 added to the PCB mix was more visible at later time points of PCB exposure. For example, Clock, Bmal1, Cry1, and Per2 were upregulated only in the PCB mix+PCB126 group after 48h exposure. Treatment with PCB126 alone decreased the expression of Cry2 and Per2 at 72h; otherwise had minimal impact on the expression of the assessed genes.

Figure 1. Time-dependent impact of PCB exposure on the expression of the core circadian rhythm genes.

Figure 1.

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Cells were exposed to PCB mix (a mix of non-coplanar PCB138, PCB153 and PCB180 in the molar ratio of 1.7:3.2:1) at 5 μM, PCB mix at 5 μM + PCB126 at 0.1%, or PCB126 at 0.1%. Control cells were treated with DMSO (vehicle) at less than 0.1%. Clock (A), Bmal1 (B), Cry1 (C), Cry2 (D), Per1 (E), Per2 (F) mRNA levels were measured by RT-qPCR. The dashed line shows the reference values for the Vehicle group. Values are mean ± SEM; n=6 in each group; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 as compared to the control group at specific time point.

We next assessed the impact of PCBs on protein levels of core circadian rhythm regulators (Figure 2). The time points that resulted in most significant changes in the gene expression were 6h and 12h (Figure 1); however, the gene expression changes are frequently more dynamic and temporary as compared to more stable protein expression alterations. Therefore, circadian proteins were analyzed at longer 12h and 24h time points. The whole cell protein was extracted, and protein expression was assessed by immunoblotting. Treatment with the PCB mix alone did not markedly affect the studied proteins; however, adding PCB126 to this mix induced a pronounced impact. For example, treatment with PCB mix + PCB126 for 12h decreased Clock, Bmal1, Per1, and Per2 protein levels (Figure 2A). Interestingly, a decrease in Bmal1 protein expression was observed only in the PCB mix + PCB126 group after 12h treatment and not in any other experimental groups. In addition, an increase in Per1 protein expression at 48h was detected only in the PCB mix + PCB126 group and not in cultures exposure to PCB mix or PCB126 alone (Figure 2B).

Figure 2. The impact of PCB exposure on the expression of circadian rhythm-related proteins.

Figure 2.

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Cells were exposed to vehicle or PCBs as in Figure 2 for 12h (A) or 24h (B) and the protein levels of Clock, Bmal1, Cry1, Cry2, Per1, and Per2 were assessed by Western blotting in the whole cell lysates. The images show representative immunoblots of the target proteins normalized to GAPDH levels. Values are mean ± SEM; n=6 in each group; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 as compared to the Vehicle group.

We also measured the impact of PCBs on mRNA and protein levels Npas2, a paralog of the Clock gene (Figure S2) and detected similar significant changes and tendencies that were found in the Clock gene expression, especially after 6h and 12h PCB exposure. The Npas2 protein expression was significantly lower at both 12h and 24h of PCB exposure.

3.2. Impact of Bmal1 silencing and/or PCB exposure on the expression of Nr1d1 and Nr1d2

Knowing that exposure to PCBs can affect the expression of core circadian regulators, we next evaluated if disruption of circadian regulation by silencing of Bmal1 can influence endothelial toxicity of PCBs. In the first series of experiments, we optimized the Bmal1 gene silencing method. Compared to non-specific siRNA, transfections of Bmal1 siRNA resulted in ~75% decrease in Bmal1 mRNA both in the vehicle group and the studied PCB exposure groups (Figure S3A), which corresponded to ~ 50% decrease in Bmal1 proteins (Figure S3B).

Bmal1 regulates expression of other circadian genes and proteins. Therefore, we analyzed the impact of Bmal1 gene disruption on the nuclear receptor subfamily 1, group D, member 1 and 2 (Nr1d1 and Nr1d2) mRNA expressions in PCB-treated brain endothelial cell cultures. Nr1d1 and Nr1d2 (Rev-Erbα and Rev-Erbβ, respectively) have overlapping functions and link circadian clock with cell metabolism (Shi et al., 2010). Consistent with the role of Bmal1 in circadian regulation, silencing of Bmal1 resulted in a significant decrease in Nr1d1 and Nr1d2 gene expression at all time points studied; i.e., at 6h, 24h, and 48h (Figures 3A, 3B, and 3C, respectively). Treatment with PCB mix and, to a lesser extent, exposure to PCB126 prevented this effect. Interestingly, a 24h exposure to a combination of PCB mix + PCB126 increased Nr1d1 and Nr1d2 expression in Bmal1-silenced hCMEC/D3 cells as compared to controls (Figures 3B and 3C).

Figure 3. Time-dependent impact of PCB exposure on Nr1d1 and Nr1d2 mRNA levels in Bmal1-silenced endothelial cells.

Figure 3.

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Cells were transfected with 75 nM Bmal1 specific or non-specific siRNA as in Figure 4, followed by exposure to PCBs as in Figure 2 for (A) 6h, (B) 24h, or (C) 48h. Values are mean ± SEM; n=3-6 in each group;*vs. non-specific siRNA Vehicle group at **p<0.01, ***p<0.001, and ****p<0.0001; #values in the Bmal1-silenced and PCB-treated groups vs. the Bmal1 silenced Vehicle group at #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001; †Values in the Bmal1-silenced and PCB-treated groups vs. the respective PCB treated group transfected with non-specific siRNA at †p<0.05 and ††p<0.01; significantly different between the PCB-treated groups transfected with non-specific siRNA at p<0.05; ‡ significantly different between the PCB-treated groups transfected with Bmal-1 siRNA at ‡‡p<0.01 and ‡‡‡‡p<0.0001.

3.3. Impact of Bmal1 silencing and/or PCB exposure on claudin-5 and occludin expression

Tight junctions (TJs) are the main structural regulator of endothelial integrity and, ultimately, the blood-brain barrier properties. Therefore, we evaluated the impact of PCBs and/or circadian disruption on the expression of TJ proteins, such as claudin-5, occludin, JAM-2, ZO-1, and ZO-2. Claudin-5, occludin, and JAM-2 are transmembrane TJ proteins, while ZO-1 and ZO-2 are TJ associated proteins that are involved in the maintenance of TJ complexes and cellular signaling (Greene et al., 2019; Otani and Furuse, 2020). Although PCBs are readily absorbed, they are slowly metabolized and even more slowly excreted. PCBs, especially the highly chlorinated congeners, tend to accumulate in lipid-rich tissues due to their lipophilic nature. Therefore, our studies on the impact of PCB exposure on TJ protein expression focused on the 24h and 48h time points.

Claudin-5 is the major TJ protein that regulates the integrity of the brain endothelium and the BBB (Hashimoto et al., 2021). Claudin-5 mRNA levels indicated time-dependent changes in response to PCB exposure. Following treatment with PCB126 alone, claudin-5 mRNA expression was not altered at a 24h time point; however, it decreased after 48h exposure (Figures 4A and 4C, left panels). Exposure to PCB mix or PCB mix + PCB126 markedly decreased claudin-5 mRNA levels after 24 and 48h as compared to vehicle treatment (Figures 4A and 4B, left panels). At the protein levels, the most pronounced change was a decrease in claudin-5 expression in the PCB mix + PCB126 treated cultures as the result of a 48h exposure (Figures 4A and 4B, right panels). In addition, the data on the impact of a 6h exposure to PCBs on claudin-5 expression is presented in Figure S4A.

Figure 4. Time-dependent impact of PCB exposure on claudin-5 and occludin mRNA and protein expression in Bmal1-silenced endothelial cells.

Figure 4.

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Cells were transfected with 75 nM Bmal1 specific or non-specific siRNA as in Supplemental Figure 3, followed by exposure to PCBs for 24h and 48h. Left panels depict changes in mRNA levels and right panels changes in protein expression of claudin-5 (A, B) and occludin (C, D). GAPDH levels were used to normalize the results. Values are mean ± SEM; n=6 in each group; *vs. non-specific siRNA Vehicle group at *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; #values in the Bmal1-silenced and PCB-treated groups vs. the Bmal1 silenced Vehicle group at ##p<0.01, ###p<0.001, and ####p<0.0001; significantly different between the PCB-treated groups transfected with non-specific siRNA at p<0.05 and ¶¶¶¶p<0.0001; ‡significantly different between the PCB-treated groups transfected with Bmal-1 siRNA at ‡p<0.05, ††p<0.01, ‡‡‡p<0.001, and ‡‡‡‡p<0.0001.

Silencing of Bmal1 markedly decreased the expression of claudin-5 mRNA after a 48h exposure as compared to cell transfected with non-specific siRNA. On the other hand, Bmal1 silencing had minimal additional impact on PCB-induced changes in claudin-5 gene or protein expression (Figures 4A and 4B).

Occludin is involved in both barrier and metabolic regulation of endothelial cells (Castro et al., 2018). Exposure to PCB mix and/or PCB126 alone for 24h or 48h did not affect occludin mRNA or protein levels (Figures 4C and 4D). However, these effects were altered by Bmal1 silencing. Indeed, exposure to PCB mix and PCB mix + PCB126 at 48h was significantly more effective in lowering occludin protein in cells with silenced Bmal1 as compared to the effects of these PCBs in cells transferred with non-specific siRNA (Figure 4D, right panel). The data on the impact of a 6h exposure to PCBs on occludin expression is presented as Figure S4B.

3.4. Bmal1 silencing alters PCB-mediated impact on o JAM-2 expression

JAM-2 is a member of the junctional adhesion molecule family and participates in interendothelial junctional complexes (Otani and Furuse, 2020). A 24h exposure to PCBs did not affect JAM-2 expression at the mRNA or protein levels, independently on PCB type and/or Bmal1 silencing (Figures 5A and 5B). On the other hand, Bmal1 silencing contributed to decreased JAM-2 protein levels in PCB mix and PCB mix + PCB126 groups after 48h exposure. This effect was not observed in cells with Bmal1 silencing and exposed to PCB126 alone, indicating specific effects of non-coplanar PCBs (Figure 5B).

Figure 5. Impact of PCB exposure on JAM-2 expression in Bmal1-silenced endothelial cells.

Figure 5.

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Cells were transfected with 75 nM Bmal1 specific or non-specific siRNA as in Figure 4, followed by exposure to PCBs as in Figure 2 for (A) 24h or (B) 48h. Left panels depict changes in mRNA levels and right panels depict changes in protein expression. GAPDH levels were used to normalize the results. Values are mean ± SEM; n=6 in each group; †values in the Bmal1-silenced and PCB-treated groups vs. the respective PCB treated group transfected with non-specific siRNA at †p<0.05.

3.5. Bmal1 silencing alters PCB-mediated impact on ZO-1 and ZO-2 expression

ZO proteins are TJ associated proteins. Silencing of Bmal1 decreased the expression of ZO-1 mRNA levels in the PCB mix + PCB126 and PCB126 groups as compared to controls after 24h exposure (Figure 6A, left panel). On the other hand, a 48h exposure to PCB126 increased ZO-1 mRNA levels both in cells transfected with Bmal1 siRNA and non-specific siRNA as compared to the respective controls (Figure 6B, left panel). ZO-1 protein levels exhibited remarkable dependency on the exposure time to PCBs. A 24h treatment with the PCB mix had a strong tendency to decrease ZO-1 protein levels as compared to control cells. In contrast, treatment with PCB mix + PCB126 for 24h markedly elevated ZO-1 protein. Silencing of Bmal1 enhanced PCB mix-induced elevation of ZO-1 protein expression at 24h (Figure 6A, right panel). In contrast, silencing of Bmal1 decreased ZO-1 protein levels in all PCB-treated groups to near non-detectable levels at a 48h time point (Figure 6B, right panel).

Figure 6. Impact of PCB exposure on ZO-1 and ZO-2 expression in Bmal1-silenced endothelial cells.

Figure 6.

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Cells were transfected with 75 nM Bmal1 specific or non-specific siRNA as in Supplemental Figure 3, followed by exposure to PCBs for 24h and 48h. Left panels depict changes in mRNA levels and right panels depict changes in protein expression of ZO-1 (A, B) and ZO-2 (C, D). GAPDH levels were used to normalize the results. Values are mean ± SEM; n=6 in each group; *vs. non-specific siRNA Vehicle group at *p<0.05, **p<0.01 and ****p<0.0001; #values in the Bmal1-silenced and PCB-treated groups vs. the Bmal1 silenced Vehicle group at #p<0.05, ##p<0.01 and ####p<0.0001; †Values in the Bmal1-silenced and PCB-treated groups vs. the respective PCB treated group transfected with non-specific siRNA at †p<0.0001; significantly different between the PCB-treated groups transfected with non-specific siRNA at ¶¶p<0.01 and ¶¶¶¶p<0.0001; ‡significantly different between the PCB-treated groups transfected with Bmal-1 siRNA at ‡‡p<0.01 and ‡‡‡‡p<0.0001.

A 24h exposure to PCBs did not affect ZO-2 expression at the mRNA levels; however, a 48h PCB126 treatment resulted in a significantly increased gene expression as compared to vehicle treatments (Figures 6C and 6D, left panels). Regarding ZO-2 mRNA and protein levels, there were no differences at 24h post PCB treatment and/or Bmal1 silencing, independent on the study group (Figure 6C, right panel). On the other hand, a 48h Bmal1 silencing contributed to a dramatic loss of ZO-2 protein in all three PCB-treated groups (Figure 6D, right panel). Overall, these effects were similar to those observed when analyzing ZO-1 protein and mRNA changes.

3.6. Impact of Bmal1 silencing and/or PCB exposure on β-catenin expression

In the last series of experiments, we measured β-catenin expression after 24h and 48h PCB exposure. β-catenin is a component of the cadherin complex and acts as an intracellular effector in the Wnt/β-catenin pathway (Pai et al., 2017). β-catenin interacts with TJ protein for a better stability of TJ complexes and is involved in cellular signaling. Analysis of mRNA expression levels did not demonstrate Bmal1 silencing-related changes after 24h or 48h PCB exposure (Figures 7A and 7B, left panels). On the protein level, no changes among treatment groups and controls were detected at the 24h time point (Figure 7A, right panel). However, silencing of Bmal1 contributed to decreased β-catenin protein levels in PCB mix and PCB mix + PCB126 treated cells as compared to controls with normal Bmal1 expression at the 48h time point (Figure 7B, right panel). A similar tendency to decrease β-catenin protein expression after Bmal1 silencing and exposure to PCB mix or PCB mix + PCB126 was observed in immunofluorescence studies (Figure 7C).

Figure 7. Impact of PCB exposure and Bmal1 silencing on β-catenin expression.

Figure 7.

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Cells were transfected with 75 nM Bmal1 specific or non-specific siRNA as in Figure 4, followed by exposure to PCBs as in Figure 2. β-catenin mRNA expression was measured 24h (A, left panel) or 48h (B, left panel) post PCB exposure, β-catenin protein expression was measured by immunoblotting 24h (A, right panel) or 48h (B, right panel) post PCB exposure and (C) by immunostaining 48h post PCB treatment. Original magnification of x100. Scale bar: 50μm. Values are mean ± SEM; n=4-6 in each group; *vs. non-specific siRNA Vehicle group at *p<0.05; #values in the Bmal1-silenced and PCB-treated groups vs. the Bmal1 silenced Vehicle group at #p<0.05 and ##p<0.01; †values in the Bmal1-silenced and PCB-treated groups vs. the respective PCB treated group transfected with non-specific siRNA at †p<0.05; significantly different between the PCB-treated groups transfected with non-specific siRNA at p<0.05 and ¶¶p<0.01.

4. Discussion

While exposure to PCBs is primarily via contaminated dietary sources or inhalation, reports indicated that natural disasters, such as hurricanes or flooding, can lead to redistribution of PCBs in the environment and enhanced human exposure. Indeed, elevated levels and bioaccumulation of PCBs in soil, and in sea animals such as mussels and bluefish, were reported as the results of hurricanes and in the vicinity of Superfund Sites (Bera et al., 2019; Deshpande et al., 2013; Mandigo et al., 2016; Smalling et al., 2016). These reports are of high significance, because the circadian rhythms and the stress response system are interconnected through oscillation of clock genes and the endocrine system, and susceptible to environmental regulation, such as those associated with natural disasters (Kawarai et al., 1997; Mukherjee et al., 2010). In addition, stress response hormones, such as glucocorticoids, can negatively influence the expression of Per (Cai et al., 2010). This strong interrelationship between the circadian rhythms and the response to stress contributes to the development of stress disorders (Jiang et al., 2011; Welcome, 2020; Yamamoto et al., 2005). We hypothesize that it may also be linked to high levels of stress during natural disasters and redistribution of PCBs as the result of these environmental events.

4.1. PCB-mediated impact on the circadian clock regulators

Important observations of the present study indicated that exposure to PCBs contributed to the alterations of rhythmicity of circadian gene expression at various time points. In addition to changes in expression of both Per1 and Per2 at the mRNA and protein levels, exposure to PCBs increased the expression of the primary clock genes, such as Clock and Bmal1. On the other hand, treatment with PCBs resulted in a decrease in the expression of circadian rhythm proteins, further indicating the disruption of circadian mechanisms. Our results indicate that there are two important time point windows, namely 6h-12h and then 48h, during which exposure to PCBs most significantly affected circadian clocks (Figure 1). The observed pattern of changes, specifically an increase in circadian gene expression and a decrease in circadian proteins due to PCB exposure, is consistent with the autoregulatory feedback mechanisms controlling circadian rhythms. Indeed, circadian regulation occurs via autoregulatory loops that control both gene expression, protein levels, protein turnover and their subcellular localization (Eide et al., 2005). On the other hand, downregulation of circadian proteins may also suggest enhanced post-translational degradation mechanisms. The obtained results are also in line with the literature reports. It was shown that exposure to ortho-substituted PCB153, which was part of the PCB mix used in the present study, can significantly alter circadian genes and behavior in the larvae zebrafish. These changes were associated with spinal deformity starting at day 7 post exposure (Aluru et al., 2020). It was also observed that the exposure to 4-hydroxy-2,3,3′,4′,5-pentachlorobiphenyl, a metabolite of PCB105 and PCB118, caused dose-dependent changes in the expression of genes regulating circadian rhythms and fatty acid metabolism in the rats (Ochiai et al., 2018b).

After establishing that exposure to PCBs alters the expression of circadian rhythm genes and proteins, we evaluated whether circadian disruption can affect cellular toxicity of PCBs. These experiments were performed upon the silencing of Bmal1, as the absence of Bmal1 results in arrhythmia in the circadian rhythms. The loss of other circadian-related genes is replaceable with their paralog partners that have similar function and structure (Abe et al., 2022; Fan et al., 2022; Haque et al., 2019). In the initial series of experiments, we focused on the impact of Bmal1 silencing on the expression of Nr1d1, also known as Rev-erbα, as another core circadian gene (Cho et al., 2012; Yin et al., 2010), which also targets a large number of genes involved in metabolism and cell survival (Cho et al., 2012; Yang et al., 2006). Nr1d1 transcription and circadian oscillation are susceptible to oxidative stress and inflammation (Yang et al., 2014). Therefore, it was interesting to demonstrate that disruption of circadian rhythms by Bmal1 silencing markedly affected the Nr1d1 and Nr1d2 gene expression levels at all time points evaluated in the present study. Interestingly, exposure to PCBs additionally affected these results. While Bmal1 silencing decreased Nr1d1 and Nr1d2 mRNA expression at 6h, 24h, and 48h, additional exposure to PCB mix or PCB mix + PCB126 increased the expression of these genes to the control values (Figure 3). This impact is difficult to explain. While treatment with PCB mix or PCB mix + PCB126 seemingly normalized Nr1d1/2 mRNA levels at 6h and 48h, it did not at 24h. Such results are consistent with the notion that exposure to PCBs can induce arrhythmicity of the circadian rhythms.

4.2. Impact of Bmal1 silencing and/or PCB exposure on tight junction proteins

Both cerebrovascular toxicity of PCBs (Kippler et al., 2016; Zhang et al., 2012), the rhythmic permeability of the blood-brain barrier (BBB) (Cuddapah et al., 2019), and the impact of circadian disruptions within BBB on neurological disorders are well recognized (Schurhoff and Toborek, 2023). The BBB is an evolutionary conserved, functional separation between circulating blood and the central nervous system (Cuddapah et al., 2019). The complex TJ system works to restrict paracellular movement of ions and solutes across the BBB. Disruption of the BBB integrity has been associated with several central nervous system pathologies (Archie et al., 2021), including several neuroinflammatory and neurodegenerative diseases, such as stroke (Brown and Davis, 2002), Alzheimer’s disease (Fiala et al., 2002), and traumatic brain injury (Morganti-Kossmann et al., 2002). The TJ system complex includes the integral transmembrane proteins, such as claudins, occludin, and JAMs, as well as cytoplasmic accessory proteins such as ZO-1 and ZO-2. Decreased expression or redistribution of TJ proteins in the brain endothelium results in the disruption of the BBB, contributes to neurotoxicity and neuroinflammation, and may facilitate cell trafficking into the brain (Weiss et al., 2009). Therefore, we evaluated claudin-5, occludin, JAM-2, ZO-1, and ZO-2 expression at different time points post PCB treatment and detected a significant fluctuation of studied TJ mRNA and/or protein levels depending on the duration of PCB exposure. Previous studies that have analyzed the effects of PCB exposure on the BBB also identified alterations of the endothelial barrier function in vitro (Hennig et al., 1999) and disruptions of TJ protein expression in brain microvessels when PCBs were administrated via oral-route (Seelbach et al., 2010). In addition, intraperitoneal injections of PCBs decreased occludin and claudin-5 mRNA level, especially in the hippocampal region of adult male rats (Selvakumar et al., 2019).

In general, non-coplanar PCB mix exhibited a more profound impact on TJ protein expression than coplanar PCB126. While a decrease in TJ protein levels was a more frequent response to PCB exposure, an increase in the expression of specific TJ proteins (e.g., ZO-1) was also assessed after treatment with non-coplanar PCB mix. This is also an important observation because overexpression of TJ proteins may have pathological outcomes, such as an adhesion-independent impact on promoting progression in cancers (Leech et al., 2015). Indeed, elevated levels of ZO-1 may play a pro-oncogenic role, as it has been described in melanoma cell lines (Smalley et al., 2005).

Silencing of Bmal1 had differential impact on PCB-mediated alterations of the expression of TJ proteins, indicating specificity of responses. For example, Bmal1 silencing markedly altered the impact of non-coplanar PCBs, but not coplanar PCB126, on occludin protein levels, especially at 48h (Figure 4D, left panel). These results confirm that non-coplanar and coplanar PCBs have different biological effects on endothelial and intestinal integrity, as observed in our previous publications (Choi et al., 2012, 2010). The most pronounced impact of Bmal1 silencing on PCB-mediated alterations of TJ protein expression was observed in the case of ZO-1 and ZO-2 protein expression. While PCB mix and PCB mix + PCB126 had no apparent effects on expression of these two TJ proteins, a concurrent silencing of Bmal1 resulted in a decrease in the expression of both ZO-1 and ZO-2 to minimal levels. Thus, the disruption of the circadian clock markedly amplified the molecular toxicity of PCBs in endothelial cells. Because TJ proteins regulate cell polarity and endothelial permeability, these effects are consistent with the disruption of the BBB and intestinal integrity, as frequently observed in PCB-exposed animals (Seelbach et al., 2010; Wahlang et al., 2016).

In the last set of experiments, we evaluated the impact of PCB exposure and circadian disruption on the expression of β-catenin. β-catenin is a member of the adherens junction family, forms the cadherin complex, and acts in the Wnt signaling pathway as an intracellular signaling transducer. It has been described previously that there is an interaction between circadian rhythm genes and Wnt/β-catenin pathway (Vallee et al., 2022), which regulates the integrity of the gut barrier function (Deaver et al., 2018; Eum et al., 2023). Our studies revealed changes in β-catenin mRNA expression 48h post PCB exposure; however, no significant changes were detected at the protein levels.

5. Conclusion

Circadian rhythms, also known as the daily dark-night cycles, play an integral role in shaping the physiology, metabolism, and behavior of all organisms. There is growing evidence that environmental pollutants disrupt circadian rhythms (Ochiai et al., 2018a; Zhao et al., 2015). In addition, shift work, stress, and poor sleep quality and quantity disrupt circadian rhythmicity (Boivin et al., 2022; Coldsnow et al., 2017; Hargreaves et al., 2022). In the current manuscript, Bmal1 gene silencing was applied for the modeling of circadian rhythm disruption, followed by exposure of endothelial cells to coplanar and non-coplanar PCBs for different exposure times. The results of the present study demonstrate that exposure to PCBs can alter the expression of circadian rhythm regulators in brain endothelial cells. Moreover, endothelial cell toxicity of PCBs shows dependency, at least in part, on circadian rhythms. Indeed, circadian dysregulation effectively potentiated the impact of PCBs on TJ protein expression, especially as the result of a prolonged 48h exposure. Overall, the results of this study suggest that brain endothelial cell metabolism can be altered by environmental toxicants, such as PCBs, by dysregulation of circadian rhythms. Our results provide new insights for toxicological studies that focus on the interactions between environmental pollutants, regulation of circadian rhythm, and the blood-brain barrier.

Supplementary Material

1

Environmental implication.

Polychlorinated biphenyls (PCBs) are widespread environmental pollutants that can be redistributed and released as the results of natural disasters, such as hurricanes. Moreover, natural disasters are associated with the disruption of circadian rhythms in affected populations. Our study indicates that exposure to PCBs markedly alters circadian rhythm regulation. Importantly, disruption of circadian rhythms potentiated endothelial toxicity of PCBs. These results indicate that circadian rhythms regulate the health impact of hazardous environmental toxicants and provide potential new direction to protect against toxicity of hazardous materials.

Highlights.

Environmental toxicants can induce brain endothelial cell dysfunction.

Exposure to PCBs alters the expression of endothelial circadian rhythm regulators.

Disruption of the Bmal1 gene enhances PCB-induced alterations of tight junctions

Circadian dysregulation potentiates endothelial toxicity of PCBs.

Funding

This work was supported by the University of Miami U-LINK Resilience Challenge grant and the funding from the National Institutes of Health (HL126559, MH128022, MH122235, MH072567, DA044579, DA050528, and ES034691).

Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Michal Toborek reports financial support, article publishing charges, and equipment, drugs, or supplies were provided by National Institutes of Health. Michal Toborek reports administrative support was provided by University of Miami School of Medicine.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

CRediT authorship Contribution statement

Timea Teglas: Conceptualization, Methodology, Formal analysis, Writing – original draft. Silvia Torices: Investigation, Visualization. Madison Taylor and Desiree Coker: Formal analysis, Resources. Michal Toborek: Methodology, Writing – review & editing, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

References

  1. Abe YO, Yoshitane H, Kim DW, Kawakami S, Koebis M, Nakao K, Aiba A, Kim JK, Fukada Y, 2022. Rhythmic transcription of Bmal1 stabilizes the circadian timekeeping system in mammals. Nat. Commun 13, 4652. 10.1038/s41467-022-32326-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aluru N, Krick KS, Mcdonald AM, Karchner SI, 2020. Developmental Exposure to PCB153 (2,2′,4,4′,5,5′-Hexachlorobiphenyl) Alters Circadian Rhythms and the Expression of Clock and Metabolic Genes. Toxicol. Sci 173, 41–52. 10.1093/TOXSCI/KFZ217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Apostoli P, Magoni M, Bergonzi R, Carasi S, Indelicato A, Scarcella C, Donato F, 2005. Assessment of reference values for polychlorinated biphenyl concentration in human blood. Chemosphere 61, 413–421. 10.1016/j.chemosphere.2005.02.034. [DOI] [PubMed] [Google Scholar]
  4. Archie SR, Al Shoyaib A, Cucullo L, 2021. Blood-Brain Barrier Dysfunction in CNS Disorders and Putative Therapeutic Targets: An Overview. Pharmaceutics 13, 1779. 10.3390/pharmaceuticsl3111779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bates MN, Buckland SJ, Garrett N, Ellis H, Needham LL, Patterson DG, Turner WE, Russell DG, 2004. Persistent organochlorines in the serum of the non-occupationally exposed New Zealand population. Chemosphere 54, 1431–1443. 10.1016/j.chemosphere.2003.09.040. [DOI] [PubMed] [Google Scholar]
  6. Bera G, Camargo K, Sericano JL, Liu Y, Sweet ST, Horney J, Jun M, Chiu W, Rusyn I, Wade TL, Knap AH, 2019. Baseline data for distribution of contaminants by natural disasters: results from a residential Houston neighborhood during Hurricane Harvey flooding. Heliyon 5, e02860. 10.1016/j.heliyon.2019.e02860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boivin DB, Boudreau P, Kosmadopoulos A, 2022. Disturbance of the Circadian System in Shift Work and Its Health Impact. J. Biol. Rhythms 37, 3–28. 10.1177/07487304211064218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brown RC, Davis TP, 2002. Calcium modulation of adherens and tight junction function: a potential mechanism for blood-brain barrier disruption after stroke. Stroke 33, 1706–1711. 10.1161/01.STR.0000016405.06729.83. [DOI] [PubMed] [Google Scholar]
  9. Cai Y, Liu S, Sothern RB, Xu S, Chan P, 2010. Expression of clock genes Per1 and Bmal1 in total leukocytes in health and Parkinson’s disease. Eur. J. Neurol 17, 550–554. 10.1111/J.1468-1331.2009.02848.X. [DOI] [PubMed] [Google Scholar]
  10. Carpenter DO, 2006. Polychlorinated biphenyls (PCBs): Routes of exposure and effects on human health. Rev. Environ. Health 21, 1–23. 10.1515/reveh.2006.21.l.l. [DOI] [PubMed] [Google Scholar]
  11. Cassin D, Dominik J, Botter M, Zonta R, 2018. PAH and PCB contamination in the sediments of the Venice Lagoon (Italy) before the installation of the MOSE flood defence works. Environ. Sci. Pollut. Res 25, 24951–24964. 10.1007/s11356-018-2524-y. [DOI] [PubMed] [Google Scholar]
  12. Castro V, Skowronska M, Lombardi J, He J, Seth N, Velichkovska M, Toborek M, 2018. Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity. J. Cereb. Blood Flow Metab 38, 317–332. 10.1177/0271678X17720816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cave M, Appana S, Patel M, Falkner KC, McClain CJ, Brock G, 2010. Polychlorinated Biphenyls, Lead, and Mercury Are Associated with Liver Disease in American Adults: NHANES 2003–2004. Environ. Health Perspect 118, 1735. 10.1289/EHP.1002720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. CDC, 2009. Fourth National Report on Human Exposure to Environmental Chemicals. Fourth Natl. Rep. Hum. Expo. to Environ. Chem 1–529. 10.15620/cdc:105345. [DOI] [Google Scholar]
  15. Cho H, Zhao X, Hatori M, Yu RT, Barish GD, Lam MT, Chong LW, Ditacchio L, Atkins AR, Glass CK, Liddle C, Auwerx J, Downes M, Panda S, Evans RM, 2012. Regulation of circadian behaviour and metabolism by REV-ERB-α and REV-ERB-β. Nature 2012 4857396. 485, 123–127. 10.1038/naturell048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Choi JJ, Choi YJ, Chen L, Zhang B, Eum SY, Abreu MT, Toborek M, 2012. Lipopolysaccharide potentiates polychlorinated biphenyl-induced disruption of the blood-brain barrier via TLR4/IRF-3 signaling. Toxicology 302, 212–220. 10.1016/j.tox.2012.08.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Choi JJ, Eum SY, Rampersaud E, Daunert S, Abreu MT, Toborek M, 2013. Exercise attenuates PCB-induced changes in the mouse gut microbiome. Environ. Health Perspect 121, 725–730. 10.1289/ehp.1306534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Choi YJ, Seelbach MJ, Pu H, Eum SY, Chen L, Zhang B, Hennig B, Toborek M, 2010. Polychlorinated Biphenyls Disrupt Intestinal Integrity via NADPH Oxidase-Induced Alterations of Tight Junction Protein Expression. Environ. Health Perspect 118, 976. 10.1289/EHP.0901751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Coldsnow KD, Relyea RA, Hurley JM, 2017. Evolution to environmental contamination ablates the circadian clock of an aquatic sentinel species. Ecol. Evol 7, 10339–10349. 10.1002/ece3.3490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Crnko S, Du Pré BC, Sluijter JPG, Van Laake LW, 2019. Circadian rhythms and the molecular clock in cardiovascular biology and disease. Nat. Rev. Cardiol 16, 437–447. 10.1038/s41569-019-0167-4. [DOI] [PubMed] [Google Scholar]
  21. Cuddapah VA, Zhang SL, Sehgal A, Sehgal A, 2019. Regulation of the Blood-Brain Barrier by Circadian Rhythms and Sleep. Trends Neurosci. 42, 500–510. 10.1016/j.tins.2019.05.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Deaver JA, Eum SY, Toborek M, 2018. Circadian Disruption Changes Gut Microbiome Taxa and Functional Gene Composition. Front. Microbiol 9, 737. 10.3389/fimicb.2018.00737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. DeBruyne JP, Weaver DR, Reppert SM, 2007. Peripheral circadian oscillators require CLOCK. Curr. Biol 17, R538–R539. 10.1016/j.cub.2007.05.067. [DOI] [PubMed] [Google Scholar]
  24. Deshpande AD, Dockum BW, Cleary T, Farrington C, Wieczorek D, 2013. Bioaccumulation of polychlorinated biphenyls and organochlorine pesticides in young-of-the-year bluefish (Pomatomus saltatrix) in the vicinity of a Superfund Site in New Bedford Harbor, Massachusetts, and in the adjacent waters. Mar. Pollut. Bull 72, 146–164. 10.1016/j.marpolbul.2013.04.008. [DOI] [PubMed] [Google Scholar]
  25. Desvignes V, Volatier JL, Bels de F., Zeghnoun A, Favrot MC, Marchand P, Bizec Le B., Rivière G, Leblanc JC, Merlo M, 2015. Study on polychlorobiphenyl serum levels in French consumers of freshwater fish. Sci. Total Environ 505, 623–632. 10.1016/j.scitotenv.2014.10.024. [DOI] [PubMed] [Google Scholar]
  26. Dirinck E, Jorens PG, Covaci A, Geens T, Roosens L, Neels H, Mertens I, Van Gaal L, 2011. Obesity and persistent organic pollutants: Possible obesogenic effect of organochlorine pesticides and polychlorinated biphenyls. Obesity 19, 709–714. 10.1038/oby.2010.133. [DOI] [PubMed] [Google Scholar]
  27. Eide EJ, Woolf MF, Kang H, Woolf P, Hurst W, Camacho F, Vielhaber EL, Giovanni A, Virshup DM, 2005. Control of Mammalian Circadian Rhythm by CKI-Regulated Proteasome-Mediated PER2 Degradation. Mol. Cell. Biol 25, 2795–2807. 10.1128/mcb.25.7.2795-2807.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Eum SY, Andras L, Hennig B, Toborek M, 2009. NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells. Toxicol. Appl. Pharmacol 240, 299–305. 10.1016/j.taap.2009.07.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Eum SY, Schurhoff N, Teglas T, Wolff G, Toborek M, 2023. Circadian disruption alters gut barrier integrity via a ß-catenin-MMP-related pathway. Mol. Cell. Biochem 478, 581–595. 10.1007/sll010-022-04536-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Fan R, Peng X, Xie L, Dong K, Ma D, Xu W, Shi X, Zhang S, Chen J, Yu X, Yang Y, 2022. Importance of Bmal1 in Alzheimer’s disease and associated aging-related diseases: Mechanisms and interventions. Aging Cell 21, e13704. 10.1111/acel.13704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Fang S, Cui Q, Dai X, 2020. Concentrations and accumulation rates of polychlorinated biphenyls in soil along an urban–rural gradient in Shanghai. Environ. Sci. Pollut. Res 27, 8835–8845. 10.1007/s11356-019-05312-w. [DOI] [PubMed] [Google Scholar]
  32. Faroon O, Ruiz P, 2016. Polychlorinated biphenyls: New evidence from the last decade. Toxicol. Ind. Health 32, 1825–1847. 10.1177/0748233715587849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Fiala M, Liu QN, Sayre J, Pop V, Brahmandam V, Graves MC, Vinters HV, 2002. Cyclooxygenase-2-positive macrophages infiltrate the Alzheimer’s disease brain and damage the blood-brain barrier. Eur. J. Clin. Invest 32, 360–371. 10.1046/J.1365-2362.2002.00994.X. [DOI] [PubMed] [Google Scholar]
  34. Fitzgerald EF, Belanger EE, Gomez MI, Hwang SA, Jansing RL, Hicks HE, 2007. Environmental exposures to polychlorinated biphenyls (PCBs) among older residents of upper Hudson River communities. Environ. Res 104, 352–360. 10.1016/j.envres.2007.01.010. [DOI] [PubMed] [Google Scholar]
  35. Gabryszewska M, Gworek B, 2020. Impact of municipal and industrial waste incinerators on PCBs content in the environment. PLoS One 15, e0242698. 10.1371/journal.pone.0242698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, Weitz CJ, 1998. Role of the CLOCK Protein in the Mammalian Circadian Mechanism. Science 280, 1564–1569. 10.1126/science.280.5369.1564. [DOI] [PubMed] [Google Scholar]
  37. Ghosh S, Murinova L, Trnovec T, Loffredo C, Washington K, Mitra P, Dutta S, 2014. Biomarkers Linking PCB Exposure and Obesity. Curr. Pharm. Biotechnol 15, 1058–1068. 10.2174/1389201015666141122203509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Glynn A, Aune M, Darnerud PO, Cnattingius S, Bjerselius R, Becker W, Lignell S, 2007. Determinants of serum concentrations of organochlorine compounds in Swedish pregnant women: a cross-sectional study. Environ. Health 6, 2. 10.1186/1476-069X-6-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Greene C, Hanley N, Campbell M, 2019. Claudin-5: gatekeeper of neurological function. Fluids Barriers CNS 16, 3. 10.1186/sl2987-019-0123-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Greig J, 1991. Halogenated biphenyls, terphenyls, naphthalenes, dibenzodioxins and related products. Food Chem. Toxicol 29, 648. 10.1016/0278-6915(91)90152-W. [DOI] [Google Scholar]
  41. Grimm F, Hu D, Kania-Korwel I, Lehmler H, Ludewig G, Hornbuckle K, Duffel M, Bergman A, Robertson L, 2015. Metabolism and metabolites of polychlorinated biphenyls (PCBs). Crit. Rev. Toxicol 45, 245. 10.3109/10408444.2014.999365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Haque SN, Booreddy SR, Welsh DK, 2019. Effects of BMAL1 manipulation on the Brain’s master circadian clock and behavior. Yale J. Biol. Med 92, 251–258. [PMC free article] [PubMed] [Google Scholar]
  43. Hargreaves JK, Oakenfull RJ, Davis AM, Pullen F, Knight MI, Pitchford JW, Davis SJ, 2022. Multiple metals influence distinct properties of the Arabidopsis circadian clock. PLoS One 17, e0258374. 10.1371/journal.pone.0258374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Hashimoto Y, Campbell M, Tachibana K, Okada Y, Kondoh M, 2012. Claudin-5: A Pharmacological Target to Modify the Permeability of the Blood-Brain Barrier. Biol. Pharm. Bull 44, 1380–1390. 10.1248/BPB.B21-00408. [DOI] [PubMed] [Google Scholar]
  45. Hennig B, Slim R, Toborek M, Robertson LW, 1999. Linoleic acid amplifies polychlorinated biphenyl-mediated dysfunction of endothelial cells. J. Biochem. Mol. Toxicol 13, 83–91. . [DOI] [PubMed] [Google Scholar]
  46. Heudorf U, Angerer J, Drexler H, 2002. Polychlorinated biphenyls in the blood plasma: current exposure of the population in Germany. Rev. Environ. Health 17, 123–134. 10.1515/REVEH.2002.17.2.123. [DOI] [PubMed] [Google Scholar]
  47. IARC, 2016. Polychlorinated Biphenyls and Polybrominated Biphenyls. IARC Monogr. Eval. Carcinog. Risks Hum 107, 9–500. [PMC free article] [PubMed] [Google Scholar]
  48. Jiang WG, Li SX, Zhou SJ, Sun Y, Shi J, Lu L, 2011. Chronic unpredictable stress induces a reversible change of PER2 rhythm in the suprachiasmatic nucleus. Brain Res. 1399, 25–32. 10.1016/J.BRAINRES.2011.05.001. [DOI] [PubMed] [Google Scholar]
  49. Kawarai T, Kawakami H, Yamamura Y, Nakamura S, 1997. Structure and organization of the gene encoding human dopamine transporter. Gene 195, 11–18. 10.1016/S0378-l119(97)00131-5. [DOI] [PubMed] [Google Scholar]
  50. Jensen AA, 1989. Background levels in humans. In: Kimbrough RD, Jensen AA (Ed.), Halogenated biphenyls, terphenyls, naphthalenes, dibenzodioxines and related products, Elsevier Science Publishers, San Diego, 348–364. [Google Scholar]
  51. Kippler M, Larsson SC, Berglund M, Glynn A, Wolk A, Åkesson A, 2016. Associations of dietary polychlorinated biphenyls and long-chain omega-3 fatty acids with stroke risk. Environ. Int 94, 706–711. 10.1016/J.ENVINT.2016.07.012. [DOI] [PubMed] [Google Scholar]
  52. Klocke C, Lein PJ, 2020. Evidence Implicating Non-Dioxin-Like Congeners as the Key Mediators of Polychlorinated Biphenyl (PCB) Developmental Neurotoxicity. Int. J. Mol. Sci 21, 1013. 10.3390/ijms21031013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Ko CH, Takahashi JS, 2006. Molecular components of the mammalian circadian clock. Hum. Mol. Genet 15, R271–R277. 10.1093/hmg/ddl207. [DOI] [PubMed] [Google Scholar]
  54. Kraft M, Rauchfuss K, Sievering S, Wöckner M, Neugebauer F, Fromme H, 2017. Quantification of all 209 PCB congeners in blood—Can indicators be used to calculate the total PCB blood load? Int. J. Hyg. Environ. Health 220, 201–208. 10.1016/j.ijheh.2016.09.006. [DOI] [PubMed] [Google Scholar]
  55. Leech AO, Cruz RGB, Hill ADK, Hopkins AM, 2015. Paradigms lost-an emerging role for over-expression of tight junction adhesion proteins in cancer pathogenesis. Ann. Transl. Med 3, 184. 10.3978/j.issn.2305-5839.2015.08.01. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Longnecker MP, Ryan JJ, Gladen BC, Schecter AJ, 2000. Correlations among human plasma levels of dioxin-like compounds and polychlorinated biphenyls (PCBs) and implications for epidemiologic studies. Arch. Environ. Health 55, 195–200. 10.1080/00039890009603406. [DOI] [PubMed] [Google Scholar]
  57. Mandigo AC, DiScenza DJ, Keimowitz AR, Fitzgerald N, 2016. Chemical contamination of soils in the New York City area following Hurricane Sandy. Environ. Geochem. Health 38, 1115–1124. 10.1007/S10653-015-9776-Y. [DOI] [PubMed] [Google Scholar]
  58. Morganti-Kossmann MC, Rancan M, Stahel PF, Kossmann T, 2002. Inflammatory response in acute traumatic brain injury: a double-edged sword. Curr. Opin. Crit. Care 8, 101–105. 10.1097/00075198-200204000-00002. [DOI] [PubMed] [Google Scholar]
  59. Mukherjee S, Coque L, Cao JL, Kumar J, Chakravarty S, Asaithamby A, Graham A, Gordon E, Enwright JF, Dileone RJ, Birnbaum SG, Cooper DC, McClung CA, 2010. Knockdown of Clock in the ventral tegmental area through RNA interference results in a mixed state of mania and depression-like behavior. Biol. Psychiatry 68, 503–511. 10.1016/J.BIOPSYCH.2010.04.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Needham LL, Barr DB, Caudill SP, Pirkle JL, Turner WE, Osterloh J, Jones RL, Sampson EJ, 2005. Concentrations of environmental chemicals associated with neurodevelopmental effects in U.S. population. Neurotoxicology 26, 531–545. 10.1016/J.NEURO.2004.09.005. [DOI] [PubMed] [Google Scholar]
  61. Ochiai M, Iida M, Agusa T, Takaguchi K, Fujii S, Nomiyama K, Iwata H, 2018. Effects of 4-Hydroxy-2,3,3 0 ,4 0 ,5-Pentachlorobiphenyl (4-OH-CB107) on liver transcriptome in rats: Implication in the disruption of circadian rhythm and fatty acid metabolism. Toxicol. Sci. 165, 118–130. 10.1093/toxsci/kfyl23. [DOI] [PubMed] [Google Scholar]
  62. Ochiai M, Iida M, Agusa T, Takaguchi K, Fujii S, Nomiyama K, Iwata H, 2018. Effects of 4-Hydroxy-2,3,30,40,5-Pentachlorobiphenyl (4-OH-CB107) on liver transcriptome in rats: Implication in the disruption of circadian rhythm and fatty acid metabolism. Toxicol. Sci 165, 118–130. 10.1093/toxsci/kfyl23. [DOI] [PubMed] [Google Scholar]
  63. Otani T, Furuse M, 2020. Tight Junction Structure and Function Revisited, Trends Cell Biol. 30, 805–817. 10.1016/J.TCB.2020.08.004. [DOI] [PubMed] [Google Scholar]
  64. Pai SG, Carneiro BA, Mota JM, Costa R, Leite CA, Barroso-Sousa R, Kaplan JB, Chae YK, Giles FJ, 2017. Wnt/beta-catenin pathway: Modulating anticancer immune response. J. Hematol. Oncol 10, 1–12. 10.1186/S13045-017-0471-6/FIGURES/3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Patterson DG, Todd GD, Turner WE, Maggio V, Alexander LR, Needham LL, 1994. Levels of non-ortho-substituted (coplanar), mono- and di-ortho-substituted polychlorinated biphenyls, dibenzo-p-dioxins, and dibenzofurans in human serum and adipose tissue. Environ. Health Perspect 102 Suppl 1, 195–204. 10.1289/EHP.94102S1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Salvo A, La Torre GL, Rotondo A, Mangano V, Gervasi T, Gervasi C, Vadalà R, Bartolomeo G, Iaria C, Lanteri G, Capparucci F, Dugo G, 2020. Accumulation of PCBs, PAHs, plasticizers and inorganic elements in Hexanchus griseus from the strait of Messina (Central Mediterranean sea). Nat. Prod. Res 34, 172–176. 10.1080/14786419.2019.1601197. [DOI] [PubMed] [Google Scholar]
  67. Schurhoff N, Toborek M, 2023. Circadian rhythms in the blood-brain barrier: impact on neurological disorders and stress responses. Mol. Brain 16, 5. 10.1186/s13041-023-00997-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Seelbach M, Chen L, Powell A, Choi YJ, Zhang B, Hennig B, Toborek M, 2010. Polychlorinated biphenyls disrupt blood-brain barrier integrity and promote brain metastasis formation. Environ. Health Perspect 118, 479–484. 10.1289/ehp.0901334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Seidl M, Le Roux J, Mazerolles R, Bousserrhine N, 2022. Assessment of leaching risk of trace metals, PAHs and PCBs from a brownfield located in a flooding zone. Environ. Sci. Pollut. Res. Int 29, 3600–3615. 10.1007/S11356-021-15491-0. [DOI] [PubMed] [Google Scholar]
  70. Selvakumar K, Bavithra S, Krishnamoorthy G, Arunakaran J, 2019. Impact of quercetin on tight junctional proteins and BDNF signaling molecules in hippocampus of PCBs-exposed rats. Interdiscip. Toxicol. 11, 294–305. 10.2478/intox-2018-0029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Sergeev AV, Carpenter DO, 2005. Hospitalization rates for coronary heart disease in relation to residence near areas contaminated with persistent organic pollutants and other pollutant. Environ. Health Perspect 113, 756–761. 10.1289/ehp.7595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Shi S, Hida A, McGuinness OP, Wasserman DH, Yamazaki S, Johnson CH, 2010. Circadian Clock Gene Bmal1 Is Not Essential After All; Functional Replacement with its Paralog, Bmal2. Curr. Biol 20, 316. 10.1016/J.CUB.2009.12.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Simhadri JJ, Loffredo CA, Trnovec T, Murinova LP, Nunlee-Bland G, Koppe JG, Schoeters G, Jana SS, Ghosh S, 2020. Biomarkers of metabolic disorders and neurobehavioral diseases in a PCB- exposed population: What we learned and the implications for future research. Environ. Res 191, 110211. 10.1016/j.envres.2020.110211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Smalley KSM, Brafford P, Haass NK, Brandner JM, Brown E, Herlyn M, 2005. Up-regulated expression of zonula occludens protein-1 in human melanoma associates with N-cadherin and contributes to invasion and adhesion. Am. J. Pathol 166, 1541–1554. 10.1016/S0002-9440(10)62370-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Smalling KL, Deshpande AD, Galbraith HS, Sharack BL, Timmons DM, Baker RJ, 2016. Regional assessment of persistent organic pollutants in resident mussels from New Jersey and New York estuaries following Hurricane Sandy. Mar. Pollut. Bull 107, 432–441. 10.1016/J.MARPOLBUL.2016.02.077. [DOI] [PubMed] [Google Scholar]
  76. Sontag N-J, Banks APW, Aylward LL, O’Rourke NA, Cavallucci DJ, Mueller JF, Drage DS, 2021. Comparison of lipid-normalised concentrations of persistent organic pollutants (POPs) between serum and adipose tissue. Int. J. Hyg. Environ. Health 236, 113801. 10.1016/j.ijheh.2021.113801. [DOI] [PubMed] [Google Scholar]
  77. Turyk M, Anderson HA, Hanrahan LP, Falk C, Steenport DN, Needham LL, Patterson DG, Freels S, Persky V, Boddy J, Budd M, Burkett M, Fiore B, Humphrey HEB, Johnson R, Lee G, Monaghan S, Reed D, Shelley T, Sonzogni W, Steele G, 2006. Wright, Relationship of serum levels of individual PCB, dioxin, and furan congeners and DDE with Great Lakes sport-caught fish consumption. Environ. Res 100, 173–183. 10.1016/J.ENVRES.2005.04.005. [DOI] [PubMed] [Google Scholar]
  78. Vallee A, Lecarpentier Y, Vallée J-N, 2022. WNT/β-catenin pathway and circadian rhythms in obsessive-compulsive disorder. Neural Regen. Res 17, 2126. 10.4103/1673-5374.332133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Van den Berg M, Birnbaum LS, Denison M, De Vito M, Farland W, Feeley M, Fiedler H, Hakansson H, Hanberg A, Haws L, Rose M, Safe S, Schrenk D, Tohyama C, Tritscher A, Tuomisto J, Tysklind M, Walker N, Peterson RE, 2006. The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci 93, 223–241. 10.1093/toxsci/kfl055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Wahlang B, Prough RA, Falkner KC, Hardesty JE, Song M, Clair HB, Clark BJ, States JC, Arteel GE, Cave MC, 2016. Polychlorinated biphenyl-xenobiotic nuclear receptor interactions regulate energy metabolism, behavior, and inflammation in non-alcoholic-steatohepatitis. Toxicol. Sci 149, 396–410. 10.1093/toxsci/kfv250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Walker WH, Walton JC, DeVries AC, Nelson RJ, 2020. Circadian rhythm disruption and mental health. Transl. Psychiatry 10, 1–13. 10.1038/s41398-020-0694-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Wang M, Safe S, Hearon SE, Phillips TD, 2019. Strong Adsorption of Polychlorinated Biphenyls by Processed Montmorillonite Clays: Potential Applications as Toxin Enterosorbents During Disasters and Floods. Environ. Pollut 255, 113210. 10.1016/J.ENVPOL.2019.113210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Wang SL, Tsai PC, Yang CY, Guo YL, 2008. Increased risk of diabetes and polychlorinated biphenyls and dioxins A 24-year follow-up study of the yucheng cohort. Diabetes Care 31, 1574–1579. 10.2337/dc07-2449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Wassermann M, Wassermann D, Cucos S, Miller HJ, 1979. World PCBs map: storage and effects in man and his biologic environment in the 1970s. Ann. N. Y. Acad. Sci 320, 69–124. 10.1111/j.1749-6632.1979.tbl3137.x. [DOI] [PubMed] [Google Scholar]
  85. Weiss N, Miller F, Cazaubon S, Couraud PO, 2009. The blood-brain barrier in brain homeostasis and neurological diseases. Biochim. Biophys. Acta 1788, 842–857. 10.1016/J.BBAMEM.2008.10.022. [DOI] [PubMed] [Google Scholar]
  86. Welcome MO, 2020. Cellular mechanisms and molecular signaling pathways in stress-induced anxiety, depression, and blood-brain barrier inflammation and leakage. Inflammopharmacology. 28, 643–665. 10.1007/S10787-020-00712-8. [DOI] [PubMed] [Google Scholar]
  87. Yamamoto T, Nakahata Y, Tanaka M, Yoshida M, Soma H, Shinohara K, Yasuda A, Mamine T, Takumi T, 2005. Acute physical stress elevates mouse period1 mRNA expression in mouse peripheral tissues via a glucocorticoid-responsive element. J. Biol. Chem 280, 42036–42043. 10.1074/JBC.M509600200. [DOI] [PubMed] [Google Scholar]
  88. Yang G, Wright CJ, Hinson MD, Fernando AP, Sengupta S, Biswas C, P. La PA, Dennery PA, 2014. Oxidative stress and inflammation modulate Rev-erbα signaling in the neonatal lung and affect circadian rhythmicity. Antioxidants Redox Signal. 21, 17–32. 10.1089/ARS.2013.5539 [DOI] [PMC free article] [PubMed] [Google Scholar]
  89. Yang X, Downes M, Yu RT, Bookout AL, He W, Straume M, Mangelsdorf DJ, Evans RM, 2006. Nuclear Receptor Expression Links the Circadian Clock to Metabolism. Cell 126, 801–810. 10.1016/J.CELL.2006.06.050. [DOI] [PubMed] [Google Scholar]
  90. Yin L, Wu N, Lazar MA, 2010. Nuclear Receptor Rev-Erbα: A Heme Receptor that Coordinates Circadian Rhythm and Metabolism. Nucl. Recept. Signal 8, nrs.08001. 10.1621/nrs.08001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Zhang B, Chen L, Choi JJ, Hennig B, Toborek M, 2012. Cerebrovascular toxicity of PCB153 is enhanced by binding to silica nanoparticles. J. Neuroimmune Pharmacol 7, 991–1001. 10.1007/S11481-012-9403-Y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  92. Zhao Y, Castiglioni S, Fent K, 2015. Environmental Progestins Progesterone and Drospirenone Alter the Circadian Rhythm Network in Zebrafish (Danio rerio). Environ. Sci. Technol 49, 10155–10164. 10.1021/acs.est.5b02226. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

1
NIHMS1899188-supplement-1.docx (3.1MB, docx)

Data Availability Statement

Data will be made available on request.

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