In vitro toxicity evaluation of single walled carbon nanotubes on human A549 lung cells

Abstract

This paper describes the in vitro cytotoxicity assessment of single walled carbon nanotubes (SWCNT) on A549 cells, a human lung cell line. Cellular viability was determined using the alamar blue (AB), neutral red (NR) and MTT assays, which evaluated metabolic, lysosomal and mitochondrial activity respectively. In addition, the total protein content of the cells was measured using the coomassie brilliant (CB) blue assay. Supernatants were also assayed for Adenylate Kinase (AK) release and Interleukin 8 (IL-8) which indicated a loss of cell membrane integrity and an inflammation response respectively. To investigate the interactions between serum components in the test medium and the test materials, exposures were conducted both in serum containing (5%) and serum-free medium. Results from the cytotoxicity tests (AB, CB, MTT) revealed the SWCNT to have very low acute toxicity to the A549 cells as all but one of the reported 24 h EC₅₀ values exceeded the top concentration tested (800 μg/ml). The SWCNT were found to interfere with a number of the dyes used in the cytotoxicity assessment and we are currently conducting a comprehensive spectroscopic study to further investigate these interactions. Of the multiple cytotoxicity assays used, the AB assay was found to be the most sensitive and reproducible. Transmission electron microscopy (TEM) studies confirmed that there was no intracellular localization of SWCNT in A549 cells following 24 h exposure; however, increased numbers of surfactant storing lamellar bodies were observed in exposed cells.

Introduction

Nanotechnology is widely perceived as one of the key technologies of the 21st century and accordingly there have been huge advances and increased funding in global technological research on nanomaterials. Single wall carbon nanotubes (SWCNT) are considered to have extensive commercial application potential due to their excellent mechanical, electrical and magnetic properties (Paradise and Goswami, in press). The broad range of increasing nanotechnology applications for SWCNT will almost certainly result in the increased potential for both human and environmental exposures to this nanomaterial. It is, therefore, imperative that toxicological research to evaluate the biocompatibility and possible adverse effects on both the health of humans and the environment is conducted concomitantly with technological research and development on nanomaterials (Dreher, 2004, Oberdörster et al., 2005, Thomas and Sayre, 2005).

Due to their size, SWCNT can easily become airborne and inhaled, hence the evaluation of their pulmonary effects has received a considerable amount of interest and a number of in vivo and in vitro studies have been performed to date. Several studies on the effects of both refined and raw CNT on the lung tissue of various animal models have been reported and there appears to be some inconsistency between the research findings (Huczko et al., 2001, Lam et al., 2004, Shvedova et al., 2005, Warheit et al., 2004). These studies highlighted the inherent difficulty in testing CNT due to their agglomerative nature in aqueous solutions; indeed some of the observed mortality was attributed to mechanical blockage of the airways resulting in asphyxiation (Warheit et al., 2004). It is now recognised that in order to elucidate the mechanisms of the pulmonary toxicity observed in these preliminary studies further more realistic in vivo inhalation studies with aerosolised SWCNT need to be conducted (Muller et al., 2006, Smart et al., 2006, Warheit, 2006).

A number of in vitro studies have also been performed on SWCNT with varying metal content and have evaluated different mechanistic endpoints. Shvedova et al. (2003) tested iron-rich (30 wt.% iron) SWCNT on human epidermal keratinocytes (HaCaT) and following 18 h exposure reported oxidative stress and loss of cell viability. They also observed that exposure resulted in ultrastructural and morphological changes in these skin cells. Recently, Kagan et al. (2006) demonstrated that iron-rich SWCNT (26 wt.% iron) resulted in a significant loss of intracellular low molecular weight thiols (GSH) and accumulation of lipid hydroperoxides in murine macrophages. Fiorito et al. (2006) investigated the effects of highly purified fullerenes and SWCNT on murine and human macrophages and found these materials did not stimulate the release of the inflammatory marker nitric oxide by murine macrophage cells in culture. In addition, they also demonstrated the uptake of each material by human macrophages to be very low and that each possessed a very low toxicity against human macrophage cells. Jia et al. (2005) exposed SWCNT (with trace amounts of metal catalysts) to alveolar macrophages isolated from guinea pigs for 6 h and found that the SWCNT elicited a more toxic response than multi walled CNT (MWCNT), quartz and fullerene. SWCNT have also been tested on human embryo kidney cells (HEK293) and were found to inhibit the proliferation of these cells by inducing cell apoptosis and decreasing cellular adhesive ability (Cui et al., 2005). As with the in vivo studies discussed earlier, differences in SWCNT toxicity and biocompatibility have also been observed with the various in vitro tests and these discrepancies can most likely be attributed to the varying percentages of catalysts and other impurities in the tested SWCNT, in addition to the different dispersion methods employed to date (Smart et al., 2006).

The objective of this study was to perform a comprehensive in vitro cytotoxicity assessment of SWCNT (10 wt.% iron) on A549 cells, a human epithelial-like lung cell line. Quartz was tested in parallel exposures to provide a benchmark of particle toxicity. As we have recently found that there was significant interaction between the SWCNT and foetal bovine serum (FBS) present in the test medium (Casey et al., in press), particle exposures were conducted both in serum containing (5%) and serum-free cell culture medium. Cytotoxicity parameters evaluated in this study, following 24 h exposure to both materials included the metabolic, lysosomal, and mitochondrial activities of the cells. In addition total protein content, cell membrane integrity and inflammation responses were also measured. The present study also employed TEM to characterise the SWCNT pre exposure, to investigate if the SWCNT were internalised by these lung cells and to examine for any ultrastructural changes in cell morphology post exposure.