Biomedical Engineering Reference
In-Depth Information
17.2 MECHANISM OF ACTION
NPs enter the cell via endocytosis and end up in lysosomes where they are exposed to hydrolytic
enzymes. Small NPs can also cross cell membranes and accumulate in specific subcellular structures
and even the nucleus. Tsoli et al. (2005) described how small NPs (1-2 nm diameter) associate with
DNA strands. But even if free NPs in cytoplasm cannot go through the nuclear membrane, they can
have eventual access to genetic material during mitosis. Thus, some NPs can penetrate the nucleus
and interfere directly with the structure and function of genomic DNA or the nuclear matrix.
The genotoxicity of NPs might also result from indirect DNA damage by the cellular production
of reactive oxygen species (ROS), the depletion of the antioxidant defense, or the altered synthesis of
DNA repair enzymes (Singh et al. 2009). ROS can be generated directly by the interaction between
the NP surface and cell membranes, alteration of mitochondrial function, or the activation of the
membrane-bound NADPH oxidase during phagocytosis (Konczol et al. 2011; Nabeshi et al. 2011;
Norppa et al. 2011; Wang et al. 2012). They are also produced in inflammatory processes induced by
the activation of neutrophils and macrophages by NPs (Greim et al. 2001; Schins 2002; Schins and
Knaapen 2007; Donaldson et al. 2010). ROS can interfere with DNA, producing different lesions
(e.g., 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-gua), single-strand breaks) or stalled replication
forks can lead to double strand breaks after replication, which can result in recombination, thus
producing permanent genome rearrangements.
NPs can exert their genotoxic effects in a variety of organs due to their capability to enter sys-
temic blood circulation. Moreover, they can cross epithelial and endothelial cell layers by transcyto-
sis and subsequently travel along the dendrites, axons, and lymphatic vessels (Durnev 2008).
A decreased antioxidant defense or DNA repair activity are two other mechanisms of action by
which an NP can induce genotoxicity, which have not yet been fully studied.
17. 3
IN VITRO
VERSUS
IN VIVO
ASSAYS, WHAT DO THEY TELL US?
The strategy to test the genotoxicity of pharmaceuticals for human use suggested in ICH S2(R1)
includes a battery of
in vitro
and
in vivo
studies (Figure 17.1); both types of assays are needed to
check different endpoints. Results obtained in
in vitro
studies require the eventual confirmation
with an
in vivo
approach since
in vitro
assays lack the complexity of an organism.
In nanogenotoxicology,
in vitro
assays are widely used and can detect different mechanisms of
action and endpoints. The localization of the NPs inside the nucleus as well as the production of
ROS will give clues to distinguishing between different mechanisms of action (Schins 2002). ROS
scavengers to check for a possible decrease in the DNA damage produced (Konczol et al. 2011;
Toduka et al. 2012; Toyooka et al. 2012) as well as inhibitors of endocytosis, nitric oxide synthase,
and cyclooxygenase-2 (Xu et al. 2009) have been used to elucidate the mechanism of oxidative
stress induced by NPs
in vitro.
Secondary inflammatory effects on DNA have to be studied
in vivo
. Relevant oxidative and
inflammatory endpoints would help in the interpretation of
in vivo
genotoxicity outcomes (Nielsen
et al. 2008; Borm et al. 2011; Downs et al. 2012).
17.4 STATE OF THE ART: STUDY OF METHODS USED IN
NANOGENOTOXICOLOGY
About 240 papers from different searches (using the terms nanogenotoxicology, genotoxicology
and NPs, genetic toxicology and NPs, and genotoxicity and NPs) in the PubMed database have been
analyzed to check the different methodologies used to evaluate the genotoxic effects of NPs until
now. Papers that involve genotoxicity studies of NPs with a potential application to nanomedicine,
as well as ones that are currently in use, have been selected to be included for the analysis. Gene
expression studies have not been included. One hundred and two papers met the criteria. Table 17.1
