Potential in vitro effects of carbon nanotubes on human aortic endothelial cells

https://doi.org/10.1016/j.taap.2009.02.018Get rights and content

Abstract

Respiratory exposure of mice to carbon nanotubes induces pulmonary toxicity and adverse cardiovascular effects associated with atherosclerosis. We hypothesize that the direct contact of carbon nanotubes with endothelial cells will result in dose-dependent effects related to altered cell function and cytotoxicity which may play a role in potential adverse pulmonary and cardiovascular outcomes. To test this hypothesis, we examined the effects of purified single- and multi-walled carbon nanotubes (SWCNT and MWCNT) on human aortic endothelial cells by evaluating actin filament integrity and VE-cadherin distribution by fluorescence microscopy, membrane permeability by measuring the lactate dehydrogenase (LDH) release, proliferation/viability by WST-1 assay, and overall functionality by tubule formation assay. Marked actin filament and VE-cadherin disruption, cytotoxicity, and reduced tubule formation occurred consistently at 24 h post-exposure to the highest concentrations [50–150 μg/106 cells (1.5–4.5 μg/ml)] for both SWCNT and MWCNT tested in our studies. These effects were not observed with carbon black exposure and carbon nanotube exposure in lower concentrations [1–10 μg/106 cells (0.04–0.4 μg/ml)] or in any tested concentrations at 3 h post-exposure. Overall, the results indicate that SWCNT and MWCNT exposure induce direct effects on endothelial cells in a dose-dependent manner.

Introduction

Engineered carbon nanomaterials, including carbon nanotubes, have elicited a significant interest due to their unique electronic and mechanical properties. However, the small size, large surface area, and high reactivity of these materials are the main factors for potential toxicity (Donaldson et al., 2006). Carbon nanotubes (CNT), including the single- and multi-walled (SWCNT and MWCNT), will have wide-spread applications in many technological fields, thus worker/consumer exposure is likely to occur, posing emerging health concerns (Donaldson et al., 2006, Maynard et al., 2006). Initial toxicological animal studies demonstrated that pulmonary deposition of SWCNT or MWCNT causes acute pulmonary inflammation as well as chronic responses such as fibrosis (Lam et al., 2004, Warheit et al., 2004, Muller et al., 2005, Shvedova et al., 2005, Mangum et al., 2006, Li et al., 2007a, Mercer et al., 2008). Furthermore, we demonstrated that CNT respiratory exposure is associated with adverse cardiovascular outcomes. Pulmonary deposition of SWCNT or MWCNT results in a rapid release of inflammatory mediators, activated blood cells, and thrombogenic proteins into the systemic circulation which may induce endothelial dysfunction (Erdely et al., 2009). Chronic SWCNT respiratory exposure triggers mitochondrial aortic alterations which may be associated with SWCNT-induced accelerated atherogenesis in apoE−/− mice, a model for human atherosclerosis (Li et al., 2007b). The CNT-related cardiovascular findings, are consistent with the current knowledge of the link between particulate matter in air pollution and the risk from cardiovascular diseases related to atherosclerosis (Brook et al., 2004).

The cardiovascular system may be affected through two main mechanisms including indirect effects, mediated by particle-induced pulmonary inflammation and dysfunction, or direct effects of particles that have traveled into the systemic circulation (Mills et al., 2007). It has been shown in animal models that some inhaled particles were located in tissues beyond the lung (Kreyling et al., 2002, Nemmar et al., 2002, Oberdorster et al., 2002). The proximity between epithelial type I and endothelial cell caveolar membrane structures might play a role in the particle translocation mechanisms (Heckel et al., 2004). The major unresolved question is whether particles translocate in sufficient numbers to exert a significant direct influence on vascular endothelial function (Mills et al., 2008a). Under both possible mechanisms of CNT-induced cardiovascular toxicity, endothelial cells are a potential target. Although many in vitro studies on CNT toxicity have been conducted (Shvedova et al., 2003, Manna et al., 2005, Monteiro-Riviere et al., 2005, Raja et al., 2007, Pacurari et al., 2008a, Pacurari et al., 2008b), the potential effects of CNT exposure on endothelial cells have not been investigated. The purpose of our studies was to evaluate potential direct toxicity of CNT on human endothelial cells, with special emphasis on the evaluation of dose-dependent effects. The dose response was select based on the hypothesis that endothelial cells are exposed to high particle concentrations at the penetration site and to low concentrations in the systemic vasculature as a result of translocation from the entrance site.

Section snippets

Reagents

Catalytically grown and purified SWCNT and MWCNT (Mitsui & Co., Ltd.) (Oberlin et al., 1976) were kindly provided by Dr. M. Endo (Shinshu University, Wakasato, Japan). Characteristics of the SWCNT (a specific surface area (BET) average 641 m2/g) and MWCNT (BET average 56 m2/g) have been described in greater detail (Kim et al., 2005, Koyama et al., 2006). Trace metal analysis by inductively coupled plasma optical emission spectrometry indicated iron content of SWCNT at 8.8% by weight and 0.27%

Cytotoxicity studies

In accordance with the recent findings on the specificity of cytotoxicity assays for CNT (Worle-Knirsch et al., 2006, Casey et al., 2008), the effects of CNT on HAEC viability was evaluated under serum-free culture conditions using both LDH and WST-1 assays. First, HAEC were exposed to SWCNT, MWCNT, or CB at a concentration of 150 μg/106 cells (4.5 μg/ml) for 3 or 24 h. The exposure of HAEC cells to either SWCNT or MWCNT resulted in elevated LDH cell release, a sign of membrane leakage, at 24 h

Discussion

The functionality of endothelial cells is fundamental for the homeostasis of the vascular system (Cai and Harrison, 2000). Due to its unique position in the vessel wall, the endothelium acts as a barrier and serves as the primary sensor for normal blood flow. The findings of this study demonstrated actin cytoskeleton disruption accompanied with altered VE-cadherin localization, reduced tubule formation, and a concomitant diminished viability of human aortic endothelial cells as a result of

Conflict of interest statement

The authors of this manuscript declare there are no conflicts of interest.

Disclaimer

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.

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    Current affiliation: National Center for Environmental Assessment, U.S. Environmental Protection Agency, Washington, DC, USA.

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