Comparative cyto-genotoxicity assessment of functionalized and pristine multiwalled carbon nanotubes on human lung epithelial cells

Abstract

Chemical functionalization extends CNT applications conferring them new functions, but could modify their toxicity. We compared cytotoxicity and genotoxic/oxidative effects of –OH functionalized and pristine MWCNTs to evaluated the influence of the functionalization exposing A549 cells to 1–40 μg/ml of both MWCNTs for 2, 4 and 24 h. Cytotoxicity was evaluated by MTT and LDH tests and apoptosis induction, direct/oxidative DNA damage by Fpg-modified comet assay. After 24 h we found viability reduction significant at 20 and 40 μg/ml for both the MWCNTs with a detectable viability reduction already at lower concentrations for MWCNTs. A significant LDH release was found only for MWCNTs. Significant apoptosis induction was found from 10 μg/ml of MWCNT-OH. A concentration-dependent increase of direct DNA damage, significant at 40 μg/ml of MWCNTs and beginning from 5 μg/ml of MWCNT-OH was detected at all exposure times. Oxidative DNA damage was not observed for both CNTs. The results indicate a different cytotoxic mechanism, by membrane damage for MWCNTs and apoptosis for MWCNT-OH, that could be explained by a different cellular uptake. Moreover, we found an earlier genotoxic effect for MWCNT-OH. The findings suggest that further studies on functionalized CNTs are necessary before using them in several applications particularly in biomedical field.

Introduction

Carbon nanotubes (CNTs), characterized by cylindrical shape and composed of carbon atoms, possess unique chemical, physical, thermal and electrical properties with potential use in various consumer, medical and industrial applications. It has been predicted that tons of carbon nanotubes will be produced worldwide every year (Maynard et al., 2006).

Multiwalled carbon nanotubes (MWCNTs) consist of several concentric graphene sheets, they are produced by high technology laboratories and are also found in particulate matter from ordinary combustion of fuel gases (Murr et al., 2004). Several studies have been performed in the last years on toxicological effects of CNTs, but existing data are controversial: fiber length and diameter, surface area, tendency to agglomerate, dispersibility in media, impurities and presence of metal catalyst due to the method of production influence toxicity and reactivity of CNTs (Donaldson et al., 2006, Herzog et al., 2007, Nel et al., 2006, Shvedova et al., 2009, Simeonova, 2009, Wang et al., 2009, Wick et al., 2007).

It is possible to increase solubility and dispersion of nanotubes by chemical treatments such as acid functionalization or other methods allowing to implement their possible applications particularly in biomedicine. In fact, functionalized CNTs were shown to cross cell membrane and deliver attached cargos into cells (Gutierrez-Praena et al., 2011). However, chemical functionalization seems to modify CNT toxic effects. In two studies chemical treatment decreased the toxicity of single-walled carbon nanotubes (SWCNTs) (Sayes et al., 2006, Zhang et al., 2007). In particular, Sayes et al. (2006) found that in human dermal fibroblasts, cytotoxicity of phenyl-SO₃H and phenyl-(COOH)₂ functionalized SWCNTs decreased as the functionalization of the tubes increased. While in a recent study that determined the influence of functionalization on SWCNT toxicity the acid-functionalized SWCNTs penetrated murine macrophages membrane, aggregated in cell cytoplasm and nuclear areas and induced enhanced toxicity (Dong et al., 2011). Another recent study that evaluated the influence of acid carboxylic functionalization of SWCNTs on human endothelial cells (HUVEC), found that the functionalization resulted in a higher toxicity compared to pristine CNTs cytotoxicity (Gutiérrez-Praena et al., 2011). In mice exposed in vivo to SWCNTs, functionalization increased pulmonary toxicity (Tong et al., 2009). Oropharyngeal aspiration of acid functionalized SWCNTs induced stronger pulmonary inflammation in mice than non-fuctionalized material (Saxena et al., 2007), suggesting that either chemical modification of the nanotubes or the degree and type of dispersion affected their toxicity (Wick et al., 2007). Tong et al., (2009) investigated cardiac toxicity in mice and found that the acid-functionalized carbon nanotubes increased cardiac ischemia/reperfusion injury evidenced by decreased recovery of cardiac function, increased infarct size and mild cardiac myofiber degeneration. While no significant ischemia/reperfusion injury was found in mice exposed to non-functionalized SWCNTs or ultrafine carbon particles.

Only a few studies evaluated biological effects of functionalized MWCNTs. Magrez et al. (2006) showed that MWCNT toxicity on human lung-tumor cell line increases when carbonyl, carboxyl and hydroxyl groups are present on their surface (Magrez et al., 2006). Coccini et al. (2010) highlighted that water solubility, high dispersibility and low agglomeration tendency, obtained by chemical functionalization with COOH and NH₂ groups were relevant in modulating cytotoxicity. In fact, they found on human astrocytes and lung epithelial cells exposed to pristine, moderately and highly functionalized MWCNTs, higher cytotoxicity effects in highly functionalized (hf) NH₂-MWCNTs. Patlolla et al. (2010a) observed that functionalized MWCNTs induce a dose-dependent toxicity on dermal fibroblast cells. In a further study of Patlolla et al. (2010b), performed on bone marrow cells of mice exposed to COOH functionalized and pristine MWCNTs, found that functionalized MWCNTs had an higher clastogenic/genotoxic potential compared to non-functionalized form. Pulmonary toxic effects caused by pristine MWCNTs and functionalized MWCNT-NH₂ or MWCNT-COOH were studied on intratracheally instilled rats (Roda et al., 2011). The results of this study showed pulmonary toxicity and inflammatory response after exposure to all the tested CNTs.

Our aim was to evaluate and compare cytotoxic and early genotoxic/oxidative effects of exposure to low concentrations (1–40 μg/ml) of commercial pristine MWCNTs and functionalized with hydroxyl group MWCNTs (MWCNT-OH) on human lung epithelial cells (A549) exposed for 2, 4 and 24 h. We specifically choose this cell line since lungs are the main potential exposure target organ during manufacture and processing of nanomaterials (Maynard et al., 2004, Shvedova et al., 2003, Smart et al., 2006) involving a large number of workers and general population. Moreover, we used low concentrations of CNTs with the attempt to reproduce the potential human exposure to such nanomaterials in different applications. We performed structural characterization of the tested carbon nanotubes using energy filtered transmission electron microscopy (EFTEM).

Our study could be useful to understand if chemical functionalization of MWCNTs, made to improve the dispersion, increases their toxicity before extend their applications particularly in the biomedical field.