Toxicological Effects of Metal Oxide Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 167

Ames test can be used for nanomaterial testing or not. In some cases, the Ames test
failed to detect genotoxicity whereas other genotoxicity tests were positive with basi-
cally the same nanomaterial. In particular, Al 2 O 3 nanoparticles did not display muta-
genic effects on cells when assessed by the Ames test, but were found to be genotoxic
when using micronucleus (MN) and comet assays in vitro as well as the MN assay
in vivo (Balasubramanyam et al. 2010; Di Virgilio et al. 2010; Balasubramanyam
et al. 2009). A possible explanation might be that larger nanoparticles are unable
to cross the bacterial cell wall, or, if they are able to cross the cell wall, they might
interfere with histidine synthesis thereby inducing false-negative or false-positive
results (Magdolenova et al. 2013). The in vitro MN assay (OECD TG487 “ In vitro
mammalian cell micronucleus test,” Adopted 22 July, 2010) detects both clastogenic
as well as aneugenic substances and is a sensitive cellular test system capable of
detecting DNA damage in mammalian cells. However, a number of studies resulted
in conflicting data, and the response depended on the cell type used, the particle
characteristics such as surface coating (Landsiedel et al. 2009; Warheit and Donner
2010). Therefore, further validation work is needed to clarify the predictive value of
the MN test for determining the genotoxicity of nanoparticles. As the time period
during which cells are exposed to nanoparticles is mostly limited to several hours,
sedimentation and uptake of particles may be a major issue.
Detecting mammalian DNA damage in vitro via the comet assay is the most
commonly used approach to assess chemical-mediated genotoxicity. However, the
mechanisms of nanoparticle-mediated genotoxicity are not yet clear. Since several
metal oxide nanoparticles have been found to enter the nucleus (Ahlinder et al. 2013),
genotoxic effects may be triggered by direct particle-DNA interactions. However,
increasing evidence also suggests that genotoxicity may result from indirect damage
via oxidative stress induced by metal oxide nanoparticles. These indirect genotoxic
effects have been further elucidated by Xu et al. (2009). They found anatase TiO 2
particles (5 nm and 40 nm) to increase the mutation rate in primary MEF with proper
dose dependence. Both particle sizes led to the formation of peroxynitrite anions and
induced deletions in the kbp range that could be protected by antioxidants. The level
of DNA damage could be reduced via suppression of cyclooxygenase-2 (COX-2).
COX-2 plays an important role in cellular inflammation and genomic instability, thus
particle-induced oxidative stress may therefore trigger COX-2 signaling pathways
(Xu et al. 2009). Since the ROS generating potential of metal oxide nanoparticles
depends on their chemical composition (Limbach et al. 2007; Kroll et al. 2011) at
least indirect genotoxicity of nanoparticles may thus be attributed to their chemistry.
The importance of size for the induction of DNA damage has been demonstrated
in the case of TiO 2 (Gurr et al. 2005) and ZnO (Hackenberg et al. 2011a). In compari-
son to particles of >200 nm, 10 and 20 nm TiO 2 induced a greater level of oxidative
DNA damage in human bronchial epithelial cells (BEAS-2B) (Gurr et al. 2005).
While bulk ZnO did not induce genotoxicity (comet assay) in nasal mucosa cells, a
significant level of DNA damage was observed for ZnO nanoparticles at a concentra-
tion of 10 µg/mL (Hackenberg et al. 2011b).
At present, it is unclear whether solubility plays an important role in nanoparti-
cle-induced genotoxicity. Sharma, Anderson, and Dhawan (2012) demonstrated that
ZnO nanoparticles were internalized by human epidermal keratinocytes and then