MECHANISMS OF RESISTANCE TO HALOGENATED AND NON-HALOGENATED AHR LIGANDS IN CHRONICALLY CONTAMINATED KILLIFISH POPULATIONS

Chronically contaminated killifish from Newark Bay (NB) NJ, and New Bedford Harbor (NBH) MA, have developed resistance to halogenated aromatic hydrocarbons that bind to and activate the aryl hydrocarbon receptor (AHR). To study the mechanisms of resistance, adult killifish were exposed to halogenated and non-halogenated AHR ligands and enzymatic and toxicological endpoints were measured in adult and embryonic fish. The chlorinated and non-chlorinated AHR ligands 3,34,4-tetrachlorobiphenyl (PCB77) and benzo-a-pyrene (B[a]P) induced cytochrome P450 1A (CYP1A) in reference site, but not in NB killifish. Expression of CYP3A (not part of the AHR gene battery) was inducible only in Flax Pond killifish. Basal expression of the phase II enzyme glutathione-s-transferase (GST) was higher in NB killifish. These results suggest that NB killifish are resistant to CYP1A induction by chlorinated and non-chlorinated AHR ligands. Higher basal GST activity observed in NB killifish could be protective against toxicity caused by contaminants found in this site. Activation of AHR and induction of CYP1A, by AHR ligands has been associated with the toxic effects caused by these chemicals. To determine the association between resistance to CYP1A induction and the toxicity caused by AHR ligands, CYP1A activity, developmental deformities and reactive oxygen species (ROS) production were measured in reference site and contaminated (NB and NBH) killifish embryos exposed to AHR ligands. 3,34,45-pentachlorobiphenyl (PCB126) and 3-methylcholantherene (3-MC) induced CYP1A, and ROS production in reference site embryos. NB and NBH embryos were resistant to PCB126 induction of CYP1A, but responded to 3-MC. Killifish embryos from NB and NBH were resistant to PCB126 induced deformities. PCB126 and 3-MC did not increase ROS production in NB or NBH killifish embryos. Alpha-naphthoflavone (ANF) (an AHR/CYP1A inhibitor) blocked PCB126 mediated deformities and CYP1A induction in reference site embryos, but increased ROS production. The P450 inhibitor, piperonyl butoxide (PBO) was able to block PCB126 mediated induction of CYP1A activity and ROS production. These results suggest that PCB126 induced deformities are dependent on activation of AHR and CYP1A induction. In chronically contaminated killifish populations, loss of sensitivity to coplanar PCBs and PAHs could be through reduced expression of AHR, or altered DNA sequence or methylation status of the CYP1A gene promoter. Hepatic AHR expression, measured by photoaffinity labeling, was lower in NB killifish than reference site animals, suggesting that NB killifish express less AHR protein. DNA sequence analysis did not reveal considerable differences between contaminated and reference site populations, however additional DNA fragments were observed in some promoters but not in others. The methylation of the CYP1A promoters was studied using methylation sensitive restriction enzymes and no differences were detected between reference site and NB killifish. Treatment with the DNA methyltransferase inhibitor AzaC did not restore CYP1A induction by PCB126 in NB killifish. These studies suggest that resistance to activation of AHR and induction of xenobiotic activating enzymes (CYP1A and CYP3A) in combination with increased expression of conjugating enzymes (GST) protects chronically contaminated killifish against these chemicals

Zinc nutritional status modulates expression of AhR-responsive P450 enzymes in vascular endothelial cells

Zinc has anti-inflammatory properties and is crucial for the integrity of vascular endothelial cells, and the development and homeostasis of the cardiovascular system. The aryl hydrocarbon receptor (AhR) which is expressed in the vascular endothelium also plays an important role in responses to xenobiotic exposure and cardiovascular development. We hypothesize that cellular zinc can modulate induction of AhR-responsive genes in endothelial cells. To determine if zinc deficiency can alter responses to AhR ligands, aortic endothelial cells were exposed to the AhR ligands 3,3′,4,4′-tetrachlorobiphenyl (PCB77) or beta-naphthoflavone (β-NF) alone or in combination with the membrane permeable zinc chelator TPEN, followed by measurements of the AhR-responsive cytochrome P450 enzymes CYP1A1 and 1B1. Compared to vehicle-treated cells, both PCB77-induced CYP1A1 activity (EROD) and mRNA expression were significantly reduced during zinc deficiency. In addition, PCB77 and β-NF-mediated up-regulation of CYP1A1 and CYP1B1 protein expression was significantly reduced in zinc-deficient endothelial cells. The inhibition of CYP1A1 and CYP1B1 protein expression caused by zinc deficiency was reversible by cellular zinc supplementation. Overall, our results strongly suggest that nutrition can modulate an environmental toxicant-induced biological outcome and that adequate levels of individual nutrients such as zinc are necessary for induction of AhR-responsive genes in vascular endothelial cells

PPARα Ligands Reduce PCB-Induced Endothelial Activation: Possible Interactions in Inflammation and Atherosclerosis

Exposure to polychlorinated biphenyls (PCBs) can activate inflammatory responses in vascular endothelial cells. Activation of peroxisome proliferator-activated receptors (PPARs) by nutrients or synthetic agonists has been shown to block pro-inflammatory responses both in vitro and in vivo. Here we demonstrate that activation of PPARα by synthetic agonists can reduce 3,3′4,4′-tetrachlorobiphenyl (PCB77)-induced endothelial cell activation. Primary vascular endothelial cells were pretreated with the PPARα ligands fenofibrate or WY14643 followed by exposure to PCB77. PPARα activation protected endothelial cells against PCB77-induced expression of the pro-inflammatory proteins vascular cell adhesion molecule-1 (VCAM-1), cycloxygenase-2 (COX-2), and PCB77-induced expression and activity of the aryl hydrocarbon receptor (AHR) responsive cytochrome P450 1A1 (CYP1A1). Furthermore, basal AHR expression was downregulated by fenofibrate and WY14643. We also investigated the possible interactions between PCBs, and basal PPAR activity and protein expression. Treatment with PCB77 significantly reduced basal mRNA expression of PPARα and the PPAR responsive gene CYP4A1, as well as PPARα protein expression. Also, PCB77 exposure caused a significant decrease in basal PPAR-dependent reporter gene expression in MCF-7 cells. Overall, these findings suggest that PPARα agonists can reduce PCB77 induction of endothelial cell activation by inhibition of the AHR pathway, and that coplanar PCB induced pro-inflammatory effects could be mediated, in part, by inhibition of PPARα expression and function

Linoleic acid induces proinflammatory events in vascular endothelial cells via activation of PI3K/Akt and ERK1/2 signaling

Linoleic acid (18:2n-6), is a major unsaturated fatty acid in the American diet. Linoleic acid is considered to be atherogenic because of its pro-oxidative and proinflammatory properties. There is substantial evidence that linoleic acid (LA) can activate vascular endothelial cells and contribute to an inflammatory response. To explore the mechanisms of LA-induced proinflammatory signaling pathways, the present study addresses the role of the phosphatidylinositol 3-kinase/amino kinase terminal (PI3K/Akt), extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways during vascular endothelial cell activation. After a 3- to 6-h exposure, LA significantly activated both Akt and ERK in endothelial cells, as assessed by western blot and immunofluorescence. In contrast, LA activated p38 MAPK already at 10 min, suggesting that p38 MAPK signaling occurred upstream of the ERK1/2 pathway. Furthermore, inhibition of ERK activity by PD98059 and PI3K/Akt activity by LY294002 or wortmannin significantly reduced the LA-induced activation of nuclear factor kappa B (NF-κB). These results suggest a contribution of both the ERK1/2 and PI3K/Akt pathways to the effect of LA on NF-κB-dependent transcription. Indeed, LA-mediated gene expression of the vascular cell adhesion molecule 1 was suppressed by PD98059, wortmannin and LY294002. These data indicate that both PI3K/Akt- and ERK1/2-mediated proinflammatory signaling events are critical in LA-induced endothelial cell activation and vascular inflammation

Quercetin blocks caveolae-dependent pro-inflammatory responses induced by co-planar PCBs

Polychlorinated biphenyls (PCBs) are widespread environmental contaminants, and co-planar PCBs can induce oxidative stress and activation of pro-inflammatory signaling cascades which are associated with atherosclerosis. The majority of the toxicological effects elicited by the co-planar PCB exposure are associated to the activation of the aryl hydrocarbon receptor (AHR) and subsequent induction of responsive genes. Previous studies from our group have shown that quercetin, a nutritionally relevant flavonoid can significantly reduce PCB77 induction of oxidative stress and expression of the AHR responsive gene cytochrome P450 1A1 (CYP1A1). We also have evidence that membrane domains called caveolae may regulate PCB-induced inflammatory parameters. Thus, we hypothesized that quercetin can modulate PCB-induced endothelial inflammation associated with caveolae. To test this hypothesis, endothelial cells were exposed to co-planar PCBs in combination with quercetin, and the expression of pro-inflammatory genes was analyzed by real-time PCR. Quercetin co-treatment significantly blocked both PCB77 and PCB126 induction of CYP1A1, vascular cell adhesion molecule 1 (VCAM-1), E-selectin and P-selectin. Exposure to PCB77 also induced caveolin-1 protein expression, which was reduced by co-treatment with quercetin. Our results suggest that inflammatory pathways induced by co-planar PCBs can be down-regulated by the dietary flavonoid quercetin through mechanisms associated with functional caveolae

Enhanced Operation of Electricity Distribution Grids Through Smart Metering PLC Network Monitoring, Analysis and Grid Conditioning

Low Voltage (LV) electricity distribution grid operations can be improved through a combination of new smart metering systems’ capabilities based on real time Power Line Communications (PLC) and LV grid topology mapping. This paper presents two novel contributions. The first one is a new methodology developed for smart metering PLC network monitoring and analysis. It can be used to obtain relevant information from the grid, thus adding value to existing smart metering deployments and facilitating utility operational activities. A second contribution describes grid conditioning used to obtain LV feeder and phase identification of all connected smart electric meters. Real time availability of such information may help utilities with grid planning, fault location and a more accurate point of supply management

Reductive Dechlorination of 3,3′,4,4′-Tetrachlorobiphenyl (PCB77) Using Palladium or Palladium/Iron Nanoparticles and Assessment of the Reduction in Toxic Potency in Vascular Endothelial Cells

Palladium-based nanoparticles immobilized in polymeric matrices were applied to the reductive dechlorination of 3,3′,4,4′-tetrachlorobiphenyl (PCB77) at room temperature. Two different dechlorination platforms were evaluated using (1) Pd nanoparticles within conductive polypyrrole films; or (2) immobilized Fe/Pd nanoparticles within polyvinylidene fluoride microfiltration membranes. For the first approach, the polypyrrole film was electrochemically formed in the presence of perchlorate ions that were incorporated into the film to counter-balance the positive charges of the polypyrrole chain. The film was then incubated in a solution containing tetrachloropalladate ions, which were exchanged with the perchlorate ions within the film. During this exchange, reduction of tetrachloropalladate by polypyrrole occurred, which led to the formation of palladium nanoparticles within the film. For the second approach, the membrane-supported Fe/Pd nanoparticles were prepared in three steps: polymerization of acrylic acid in polyvinylidene fluoride microfiltration membrane pores was followed by ion exchange of Fe 2+ , and then chemical reduction of the ferrous ions bound to the carboxylate groups. The membrane-supported iron nanoparticles were then soaked in a solution of tetrachloropalladate resulting in the deposition of Pd on the Fe surface. The nanoparticles prepared by both approaches were employed in the dechlorination of PCB77. The presence of hydrogen was required when the monometallic Pd nanoparticles were employed. The results indicate the removal of chlorine atoms from PCB77, which led to the formation of lower chlorinated intermediates and ultimately biphenyl. Toxicity associated with vascular dysfunction by PCB77 and biphenyl was compared using cultured endothelial cells. The data strongly suggest that the dechlorination system used in this study markedly reduced the proinflammatory activity of PCB77, a persistent organic pollutant

Coplanar polychlorinated biphenyl-induced CYP1A1 is regulated through caveolae signaling in vascular endothelial cells

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that can induce inflammatory processes in the vascular endothelium. We hypothesize that the plasma membrane microdomains called caveolae are critical in endothelial activation and toxicity induced by PCBs. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. We focused on the role of caveolae and their major protein component, caveolin-1 (Cav-1), on aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 1A1 (CYP1A1) by coplanar PCBs. Endothelial cell exposure to PCB77 increased both caveolin-1 and CYP1A1 levels in a time-dependent manner in total cell lysates, with a maximum increase at 6 h. Furthermore, PCB77 accumulated mainly in the caveolae-rich fraction, as determined by gas chromatograph–mass spectrometry. Immunoprecipitation analysis revealed that PCB77 increased AhR binding to caveolin-1. Silencing of caveolin-1 significantly attenuated PCB77-mediated induction of CYP1A1 and oxidative stress. Similar effects were observed in caveolin-1 null mice treated with PCB77. These data suggest that caveolae may play a role in regulating vascular toxicity induced by persistent environmental pollutants such as coplanar PCBs. This may have implications in understanding mechanisms of inflammatory diseases induced by environmental pollutants

Alumina nanoparticles induce expression of endothelial cell adhesion molecules

Nanotechnology is a rapidly growing industry that has elicited much concern because of the lack of available toxicity data. Exposure to ultrafine particles may be a risk for the development of vascular diseases due to dysfunction of the vascular endothelium. Increased endothelial adhesiveness is a critical first step in the development of vascular diseases, such as atherosclerosis. The hypothesis that alumina nanoparticles increase inflammatory markers of the endothelium, measured by the induction of adhesion molecules as well as the adhesion of monocytes to the endothelial monolayer, was tested. Following characterization of alumina nanoparticles by transmission electron microscopy (TEM), electron diffraction, and particle size distribution analysis, endothelial cells were exposed to alumina at various concentrations and times. Both porcine pulmonary artery endothelial cells and human umbilical vein endothelial cells showed increased mRNA and protein expression of VCAM-1, ICAM-1, and ELAM-1. Furthermore, human endothelial cells treated with alumina particles showed increased adhesion of activated monocytes. The alumina particles tended to agglomerate at physiological pH in serum-containing media, which led to a range of particle sizes from nano to micron size during treatment conditions. These data show that alumina nanoparticles can elicit a proinflammatory response and thus present a cardiovascular disease risk

Induction of gene pattern changes associated with dysfunctional lipid metabolism induced by dietary fat and exposure to a persistent organic pollutant

Environmental modulators of chronic diseases can include nutrition, lifestyle, as well as exposure to environmental toxicants such as persistent organic pollutants. A study was designed to explore gene expression changes as affected by both dietary fat and exposure to the polychlorinated biphenyl PCB77. Mice were fed for 4 months diets enriched with high-linoleic acid oils (20% and 40% as calories), and during the last 2 months half of each group was exposed to PCB77. Ribonucleic acids (RNA) were extracted from liver tissue to determine gene expression changes using DNA microarray analysis. Our microarray data demonstrated a significant interaction between dietary fat and PCB exposure. Deregulated genes were organized into patterns describing the interaction of diet and PCB exposure. Annotation of the deregulated genes matching these probe sets revealed a significant high-fat mediated induction of genes associated with fatty acid metabolism, triacylglycerol synthesis and cholesterol catabolism, which was down-regulated in animals exposed to PCB77. Many of these genes are regulated by the peroxisome proliferator activated receptor-α (PPARα), and changes in PPARα gene expression followed the same gene pattern as described above. These results provide insight into molecular mechanisms of how dietary fat can interact with environmental pollutants to compromise lipid metabolism