Carboxylated Short Single-Walled Carbon Nanotubes But Not Plain and Multi-Walled Short Carbon Nanotubes Show in vitro Genotoxicity

Abstract

Long carbon nanotubes (CNTs) resemble asbestos fibers due to their high length to diameter ratio and they thus have genotoxic effects. Another parameter that might explain their genotoxic effects is contamination with heavy metal ions. On the other hand, short (1-2 µm) CNTs do not resemble asbestos fibers, and, once purified from contaminations, they might be suitable for medical applications. To identify the role of fiber thickness and surface properties on genotoxicity, well-characterized short pristine and carboxylated single-walled (SCNTs) and multi-walled (MCNTs) CNTs of different diameters were studied for cytotoxicity, the cell's response to oxidative stress (immunoreactivity against hemoxygenase 1 and glutathione levels), and in a hypoxanthine guanine phosphoribosyltransferase (HPRT) assay using V79 chinese hamster fibroblasts and human lung adenocarcinoma A549 cells. DNA repair was demonstrated by measuring immunoreactivity against activated histone H2AX protein. The number of micronuclei as well as the number of multinucleated cells was determined. CNTs acted more cytotoxic in V79 than in A549 cells. Plain and carboxylated thin (<8 nm) SCNTs and MCNTs showed greater cytotoxic potential and carboxylated CNTs showed indication for generating oxidative stress. Multi-walled CNTs did not cause HPRT mutation, micronucleus formation, DNA damage, interference with cell division, and oxidative stress. Carboxylated, but not plain, SCNTs showed indication for in vitro DNA damage according to increase of H2AX-immunoreactive cells and HPRT mutation. Although short CNTs presented a low in vitro genotoxicity, functionalization of short SCNTs can render these particles genotoxic.

Evaluating the mechanistic evidence and key data gaps in assessing the potential carcinogenicity of carbon nanotubes and nanofibers in humans

“…Exposure to SWCNTs has been associated with increased levels of double strand breaks (measured by γH2AX immunostaining) in human lymphocytes (Cveticanin et al 2010) and A549 cells (Mrakovcic et al 2015). Another study showed unaltered levels of γH2AX immunostaining in human mesothelial cells, although the levels were regarded to be nominally increased (approximately 1.2-fold) (Pacurari et al.…”

“…In addition, 13 out of 18 studies report increased micronuclei frequency in human cell lines after exposure to MWCNTs, including three independent studies of MWCNT-7 (Muller et al 2008a, 2008b; Asakura et al 2010; Cveticanin et al 2010; Migliore et al 2010; Di Giorgio et al 2011; Srivastava et al 2011; Kato et al 2013; Wu et al 2013; Darne et al 2014; Tavares et al 2014; Visalli et al 2015; Kim et al 2016); five studies showed null effects (Szendi & Varga 2008; Thurnherr et al 2011; Lindberg et al 2013; Ponti et al 2013; Mrakovcic et al 2015). Eight out of 13 studies report increased micronuclei frequency in human cell lines after exposure to either SWCNTs or MWCNTs (Cveticanin et al 2010; Migliore et al 2010; Cicchetti et al 2011; Di Giorgio et al 2011; Kisin et al 2011; Manshian et al 2013; Darne et al 2014; Kim & Yu 2014; Kim et al 2016); five studies showed null effects (Kisin et al 2007; Lindberg et al 2009; Lindberg et al 2013; Pelka et al 2013; Mrakovcic et al 2015). Null effect studies used CNTs similar to the CNTs that caused micronuclei formation with respect to diameter, length, specific surface area and purity, although it should be emphasized that many publications contain insufficient information on fiber characteristics.…”

“…MWCNTs with structural defects increased micronuclei frequency, whereas heated and ground MWCNTs were not as genotoxic in regards to micronuclei formation (Muller et al 2008b). A number of studies have shown unaltered levels of micronuclei after MWCNT exposure in lung epithelial A549 cells (2–5 μm, Thurnherr et al 2011; 0.5–2 μm, Mrakovcic et al 2015), BEAS-2B (1–5 μm, Lindberg et al. 2013), Chinese hamster V79 fibroblasts (0.5–2 μm, Mrakovcic et al 2015), and murine fibroblasts (1.5 μm, Ponti et al 2013).…”

Effect of surface functionalizations of multi-walled carbon nanotubes on neoplastic transformation potential in primary human lung epithelial cells

“…In vitro administered fSWCNTs and fMWCNTs with strong negative charge also increased genotoxicity, hampered natural killer cell function, and inhibited cell division. Of note, an increasing number of recent studies show that fCNTs exhibit enhanced genotoxicity and apoptosis induction compared to parent CNTs, but the opposite for cellular damage and cytotoxicity assays, possibly due to uptake route (Alam et al, 2016; Mrakovcic et al, 2015; Ursini et al, 2012). …”

Safety assessment of nanoparticles for drug delivery by means of classic in vitro assays and beyond

“…In an extensive study Mrakovcic et al [95] Among metallic nanoparticles, gold nanoparticles (AuNPs) are the most promising and most widely used systems used for therapeutic, diagnostic or 'theranostic' applications. Boisselier et al [96] (2009) provides a good overview of the preparation, the field of applications and the cytotoxicity of AuNPs.…”