PCB 77 dechlorination products modulate pro-inflammatory events in vascular endothelial cells

Abstract

Persistent organic pollutants such as polychlorinated biphenyls (PCBs) are associated with detrimental health outcomes including cardiovascular diseases. Remediation of these compounds is a critical component of environmental policy. Although remediation efforts aim to completely remove toxicants, little is known about the effects of potential remediation byproducts. We previously published that Fe/Pd nanoparticles effectively dechlorinate PCB 77 to biphenyl, thus eliminating PCB-induced endothelial dysfunction using primary vascular endothelial cells. Herein, we analyzed the toxic effects of PCB congener mixtures (representative mixtures of commercial PCBs based on previous dechlorination data) produced at multiple time points during the dechlorination of PCB 77 to biphenyl. Compared with pure PCB 77, exposing endothelial cells to lower chlorinated PCB byproducts led to improved cellular viability, decreased superoxide production, and decreased nuclear factor kappa B activation based on duration of remediation. Presence of the parent compound, PCB 77, led to significant increases in mRNA and protein inflammatory marker expression. These data implicate that PCB dechlorination reduces biological toxicity to vascular endothelial cells.

Fig. 1

Dechlorination of PCB 77 alters cellular oxidative stress. Cells grown on chamber slides were treated with DMSO (vehicle control) or dechlorination products with a total concentration of 5 μM total concentrations and were incubated for 4 h. Cells were then treated with N, N′-(1,2-dihydroxyethylene) bisacrylamide (DHE, in red) for superoxide detection. Slides were fixed with DAPI (in blue) for nuclear staining; a fluorescent images were recorded at 40× and b DHE fluorescence was quantified and normalized to DAPI

Fig. 2

Dechlorination of PCB 77 attenuates NFκB activation. Cells were treated with vehicle control (DMSO), 0 (PCB 77, 5 μM) 10, 24, and 48 h dechlorination mixtures (5 μM) and incubated for 4 h. Both PCB 77 and dechlorination products (10 and 24 h product treatments) significantly increased DNA binding of NFκB over DMSO and biphenyl (48 h treatment). Results are given as mean±SEM. Different statistical marker letters represent significant differences among treatment groups (determined by P_value≤0.05)

Fig. 3

Dechlorination products alter CYP1A1 (a), MCP-1 (b), and VCAM-1 (c) mRNA expression. The presence of PCB 77 significantly upregulated mRNA inflammatory markers in ECs; inflammation decreased with further dechlorination of the parent compound. Please see Table 2 for detailed treatment descriptions. The vehicle, DMSO (treatment 1), was applied at 0.1 %v/v for all treatments. Treatments representing the dechlorination products at 0 (treatment 2), 10 (treatment 3), 24 (treatment 5), and 48 h (treatment 8) are the same as those tested previously for NFκB, superoxide production, and viability. Treatments 4 and 6 excluded PCB 77 while maintaining the representative concentrations of the dechlorination products found at 10 and 24 h, respectively. In contrast, treatments 9 and 10 contained only PCB 77 at the concentrations found in the 10 and 24 h mixtures, respectively. Treatment 7 consisted of the 24 h dechlorination mixture with the level of PCB 77 found in the 10 h mixture. Different statistical marker letters represent significant differences among treatment groups (determined by P_value≤0.05). For example, in CYP1A1, treatment 2 (statistical marker “b”) is significantly different from treatment 1 (“a”) and treatment 4 (“c”)

Fig. 4

Dechlorination mixtures alter CYP1A1 (a), MCP-1 (b), and VCAM-1 (c) protein expression. The presence of PCB 77 significantly upregulated protein inflammatory markers in ECs; inflammation decreased with further dechlorination of the parent compound. Treatments are the same as described in Fig. 3 (please see Table 2 for detailed treatment descriptions). Statistical differences among treatment groups (determined by P_value≤0.05) are represented by marker letters, as stated previously

Fig. 5

PCB 77 increases ROS production and downstream cellular dysfunction. PCB 77 interacts with caveolae in the plasma membrane, is endocytosed into the cell, and interacts with the AhR and its chaperone ARNT (aryl hydrocarbon receptor nuclear translocater), leading to nuclear translocation, XRE (xenobiotic response element) binding and CYP1A1 upregulation. CYP1A1 protein uncoupling occurs during PCB 77 metabolism and leads to an increase in cellular ROS. NFκB is activated in response to the change in cellular redox status and translocates to the nucleus where it acts as a transcription factor to upregulate adhesion molecules and cytokines