Toxicological Effects of Metal Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 197

damage in human fetal lung fibroblasts (MRC-5). When the fibroblasts were exposed
to 1 nM gold nanoparticles, a significant increase in 8 hydroxydeoxyguanosine
(8OHdG), a marker of oxidative stress, was found. In addition, they detected a down-
regulation of DNA repair genes suggesting that gold nanoparticles at a concentration
of 1 nM may interact directly or indirectly with regulators of genomic integrity (Li
et al. 2008). A comparative proteomic analysis by two-dimensional gel electropho-
resis further demonstrated the upregulation of a range of oxidative stress-related pro-
teins. The results obtained from the comet assay confirmed DNA damage in MR-5
fibroblasts after treatment with 1 nM gold nanoparticles (Li et al. 2011).
In a study characterizing size-dependency of copper nanoparticles, Midander
et al. reported that nanosized copper particles (100 µm, 80 µg/mL) caused a higher
degree of DNA damage (comet assay) in human lung cells (A549) than their micro-
sized counterparts (Midander et al. 2009). The effect seemed to be predominately
mediated by particles because no DNA damage was induced when using the frac-
tion containing only released copper. These findings suggest that the genotoxic-
ity of copper nanoparticles is indeed particle related rather than being caused by
released metal ions, and also that genotoxicity of the nanomaterials is dependent on
the particle size.
As mentioned earlier, platinum nanoparticles were found to be of low or no cyto-
toxicity. However, in some reports, genotoxic behavior of these metal nanoparticles
has been described. For instance, platinum nanoparticles of 5-8 nm in size were
found to induce p53 activation and DNA damage in human cells (Asharani et al.
2010). Comparing platinum nanoparticles of different sizes, Pelka et al. could cor-
relate an impairment of DNA integrity with particle size in an inverse manner. The
smaller particles (<20 nm and <100 nm) displayed stronger DNA damage than their
micro-sized counterparts (<100 nm). Because ROS formation could not be observed,
a contribution of oxidative stress to the DNA-damaging properties of the nanoscaled
platinum particles could be virtually excluded (Pelka et al. 2009). This demonstrates
the need for investigating different toxicological end points to obtain reliable infor-
mation on nanoparticle toxicity.
In the nanoGEM project, we analyzed Ag50.PVP and Ag200.PVP for their ability
to induce chromosome breakage, aneuploidy, or point mutagenicity in the absence or
presence of an extrinsic metabolizing system using the micronucleus assay and the
Ames test, respectively. This was the first study in which the micronucleus assay on
V79 cells and the Ames test were performed according to OECD guidelines (AMES:
OECD 471, MN: OECD 487) considering the adaptation of the testing guidelines to
manufactured nanomaterials in particular elements. In addition, the comet assay was
performed using 3D bronchial models (EpiAirway ™ , MatTek Corp., Ashland, USA).
All Ames tests performed with silver nanoparticles have been negative so far.
The results obtained in the nanoGEM project with the micronucleus test revealed
increased genotoxic effects in V79 cells when silver nanoparticles were used at con-
centrations >11 µg/mL (Ag50.PVP) and >27.5 µg/mL (Ag200.PVP), respectively
(Table 9.1). Testing of supernatants of suspended Ag200.PVP and Ag50.PVP nano-
materials after high-speed centrifugation also showed increased genotoxic effects in
V79 cells. This suggests that free PVP or silver ions, released from the nanomaterial,
contribute to the increased toxicity of silver nanoparticles.