Cerebrovascular toxicity of PCB153 is enhanced by binding to silica nanoparticles

Abstract

Environmental polychlorinated biphenyls (PCBs) are frequently bound onto nanoparticles (NPs). However, the toxicity and health effects of PCBs assembled onto nanoparticles are unknown. The aim of this study was to study the hypothesis that binding PCBs to silica NPs potentiates PCB-induced cerebrovascular toxicity and brain damage in an experimental stroke model. Mice (C57BL/6, males, 12-week-old) were exposed to PCB153 bound to NPs (PCB153-NPs), PCB153, or vehicle. PCB153 was administered in the amount of 5 ng/g body weight. A group of treated animals was subjected to a 40 min ischemia, followed by a 24 h reperfusion. The blood-brain barrier (BBB) permeability, brain infarct volume, expression of tight junction (TJ) proteins, and inflammatory mediators were assessed. As compared to controls, a 24 h exposure to PCB153-NPs injected into cerebral vasculature resulted in significant elevation of the BBB permeability, disruption of TJ protein expression, increased proinflammatory responses, and enhanced monocyte transmigration in mouse brain capillaries. Importantly, exposure to PCB153-NPs increased stroke volume and potentiated brain damage in mice subjected to ischemia/reperfusion. A long-term (30 days) oral exposure to PCB153-NPs resulted in a higher PCB153 content in the abdominal adipose tissue and amplified adhesion of leukocytes to the brain endothelium as compared to treatment with PCB153 alone. This study provides the first evidence that binding to NPs increases cerebrovascular toxicity of environmental toxicants, such as PCB153.

Figure 1. Treatment with PCB153-NPs disrupts expression of tight junction proteins and BBB integrity

Mice were exposed to PCB153-NPs by infusion into the internal carotid artery (ICA) at the dose of 5 ng PCB153/g body weight bound to 1.04 × 10⁵ silica NPs. Control mice were infused with the same amounts of NPs, PCB153 dissolved in DMSO, or vehicle (PBS or 0.01% DMSO). Brain microvessels were isolated 24 h post treatment and analyzed for occludin (A) and claudin-5 (B) expression by immunoblotting and immunofluorescence. Immunoreactivity of occludin and claudin-5 was stained in green and red, respectively; scale bar=20 μm. The blots in A and B are representative images from all experiments and the quantified results are depicted in the form of bar graphs. Arrows indicate disrupted continuity of tight junction proteins. Results are mean ± SEM, n=4. *Significantly different as compared to control groups at *p<0.05 or ***p<0.001. ^†Results in the PCB153-NP group are statistically different from those in the PCB153 group at ^†p<0.05 or ^†††p<0.001.

Figure 1. Treatment with PCB153-NPs disrupts expression of tight junction proteins and BBB integrity

Mice were exposed to PCB153-NPs by infusion into the internal carotid artery (ICA) at the dose of 5 ng PCB153/g body weight bound to 1.04 × 10⁵ silica NPs. Control mice were infused with the same amounts of NPs, PCB153 dissolved in DMSO, or vehicle (PBS or 0.01% DMSO). Brain microvessels were isolated 24 h post treatment and analyzed for occludin (A) and claudin-5 (B) expression by immunoblotting and immunofluorescence. Immunoreactivity of occludin and claudin-5 was stained in green and red, respectively; scale bar=20 μm. The blots in A and B are representative images from all experiments and the quantified results are depicted in the form of bar graphs. Arrows indicate disrupted continuity of tight junction proteins. Results are mean ± SEM, n=4. *Significantly different as compared to control groups at *p<0.05 or ***p<0.001. ^†Results in the PCB153-NP group are statistically different from those in the PCB153 group at ^†p<0.05 or ^†††p<0.001.

Figure 2. Treatment with PCB153-NPs increases permeability of the BBB

Mice were injected with vehicle, PCB153 and/or NPs as in Figure 1. BBB permeability was evaluated using sodium fluorescein 24 h post treatment. Results are mean ±SEM, n=4. ***Significantly different as compared to other groups at p<0.001.

Figure 3. Exposure to PCB153-NPs potentiates inflammatory responses in brain capillaries

Mice were treated as in Figure 1, followed by brain microvessel isolation. mRNA levels of TNF-α (A), IL-1β (B), ICAM-1 (C), and VCAM-1 (D) were determined by real-time PCR. Results are mean ± SEM, n=6. *Significantly different as compared to control groups at **p<0.01 or ***p<0.001. ^†Results in the PCB153-NPs group are statistically different from those in the PCB153 group at ^†p<0.05, ^††p<0.01, or ^†††p<0.001.

Figure 4. Treatment with PCB153-NPs enhances monocyte transmigration

(A) Mice were exposed to vehicle, PCB153, and/or NPs as in Figure 1, followed by injection with monocytic J774.1 cells labeled with CFDA-SE (green) into the internal carotid artery. In addition, brain sections were stained for claudin-5 (red) to visualize the vessels. Closed arrowheads indicate labeled J774.1 cells inside cerebral vessels, while open arrowheads indicate labeled J774.1 cells that appear to be present in the perivascular space. Scale bar = 20 μm. (B) Con uent hCMEC/D3 cells cultured on Transwell inserts were treated with PCB153-NPs (PCB153, 1.6 μM; NPs, 2.08 × 10⁵), PCB153 (1.6 μM), NPs (2.08 × 10⁵), or vehicle for 24 h. THP-1 monocytic cells were labeled with calcein AM, added on the top of endothelial monolayers, and their transendothelial migration was assessed 4 h later. Data are mean ± standard deviation (SD), n=6. *Significantly different as compared to control groups at p<0.05.

Figure 4. Treatment with PCB153-NPs enhances monocyte transmigration

(A) Mice were exposed to vehicle, PCB153, and/or NPs as in Figure 1, followed by injection with monocytic J774.1 cells labeled with CFDA-SE (green) into the internal carotid artery. In addition, brain sections were stained for claudin-5 (red) to visualize the vessels. Closed arrowheads indicate labeled J774.1 cells inside cerebral vessels, while open arrowheads indicate labeled J774.1 cells that appear to be present in the perivascular space. Scale bar = 20 μm. (B) Con uent hCMEC/D3 cells cultured on Transwell inserts were treated with PCB153-NPs (PCB153, 1.6 μM; NPs, 2.08 × 10⁵), PCB153 (1.6 μM), NPs (2.08 × 10⁵), or vehicle for 24 h. THP-1 monocytic cells were labeled with calcein AM, added on the top of endothelial monolayers, and their transendothelial migration was assessed 4 h later. Data are mean ± standard deviation (SD), n=6. *Significantly different as compared to control groups at p<0.05.

Figure 5. Exposure to PCB153-NPs increases the infarct volume in the experimental stroke model

Mice were treated as in Figure 1 and subjected to a 40 min MCAO, followed by a 24 h reperfusion, and staining with 2,3,5-triphenyltetrazolium chloride (TTC) to visualize viable tissue. Unstained area (arrows) corresponds to damaged brain tissue. Quantified results of negative TTC staining are depicted in the form of bar graphs. Results are mean ± SEM, n=5–7. ***Significantly different as compared to other groups at p<0.001.

Figure 6. Long-term exposure to PCB153-NPs potentiates PCB153 adipose accumulation and leukocyte attachment to cerebral vessels

(A) Mice were exposed orally to PCB153-NPs (5 ng PCB153/g body weight bound to 1.04 × 10⁵ silica NPs), the equimolar dose of PCB153, or the appropriate vehicles for 30 days. PCB153 levels were assessed in adipose tissue. Results are mean ± SEM, n=5–6. *Significantly different as compared to other groups at p<0.05. (B) Mice were exposed as in (A), followed by installation of the cranial window. Circulating leukocytes were fluorescently labeled by i.p. injection with rhodamine 6G and the interactions of labeled leukocytes with the brain endothelium were detected via cranial window under fluorescent microscope. Arrows indicate leukocytes inside the cerebral vessels that appear to be attached to the brain endothelium. Scale bar = 20 μm.