Biomedical Engineering Reference
In-Depth Information
in the genotoxicity testing of novel chemicals and pharmaceuticals, as well as nanoparticles. It is
called “Comet” because the microscopic detection of damaged DNA fragments in individual cells
appears as comets upon cell lysis and subsequent DNA denaturation and electrophoresis. Kim et al.
(2011) have found that BEAS-2B cells treated with Ag NPs exhibited a dose-dependent increase of
DNA breakage by the Comet assay. In addition, there is a review that concludes at least 46 cellular,
in vitro
studies using the Comet assay (Karlsson, 2010). The major disadvantage of this assay is that
it does not measure fixed mutations.
The micronucleus (MN) assay is one of the most successful and reliable tests for genotoxic carcin-
ogens, and is quite appropriate for the toxicological screening of potential genotoxic compounds. The
basis of this assay is the microscopic detection of a cell's chromosome or chromosome fragment that
has failed to integrate into the nucleus of its daughter cell after division. Chromosomal breakages in
Ag NP-treated cells are found by the MN assay (AshaRani et al., 2008). And there are several other
types of nanomaterials proven to induce a significant increase of MN frequencies. The MN assay
can detect both chromosomal and genomic mutations, while it can only be applied to dividing cells.
The Ames test (or bacterial reversion mutation test) is usually used as an adjunct technique. The
test is performed in prokaryotic systems, so it is complicated to translate to a eukaryotic system.
Moreover, some nanoparticles might not be able to cross the bacterial cell wall or they might only
be harmful to bacteria.
10.3.7 I
NflaMMatIoN
a
ssay
Inflammation involves the complex interactions of multiple cell types, so it is only possible to mea-
sure the markers of proinflammatory signaling and gene expression
in vitro
. The most common
technique is to measure the cytokines and/or chemokines produced by cells via enzyme-linked
immunosorbent assay (ELISA). The release of inflammation markers (IL1α, IL1β, IL6, IL10,
MIP1α, MIP1β, MIP2, GCSF, and TNFα) were measured in RAW 246.7 cells exposed to various
sized Ag-NPs. All markers were induced the most by 20 nm Ag-NPs (Park et al., 2011).
10.3.8 a
dvaNtage
aNd
d
IsadvaNtage
of
I
n
V
Itro
a
ssay
Cell-culture studies are practical approaches in understanding how an agent will react in the body.
They can reveal the primary effects of target cells in the absence of secondary effects caused by
inflammation, and the identification of the primary mechanisms of toxicity in the absence of the
physiological and compensatory factors that confound the interpretation of whole
in vivo
studies
(Arora et al., 2012).
Compared to animal studies, they are easier to control, perform, interpret, and reproduce.
Controlling the experimental condition is crucial since all the other factors, except nanoparticles,
causing cell death should be minimized or eliminated. Besides,
in vivo
toxicity studies have rela-
tively lower costs and faster speeds and, more importantly, they arise less ethical issues.
In vitro
systems are simple when only one cell line is tested, and the complexity of these
in vitro
systems
increases when multiple cell types are included with the purpose to better mimic the
in vivo
envi-
ronment. The co-culture of different types of cells, for example dendritic cells and epithelial cells,
are cultured together to mimic the lung surface (Rothen-Rutishauser et al., 2005), possibly generates
data with more meaning but is different to interpret.
I nevit ably,
in vitro
systems have a bunch of disadvantages. The main shortcoming is that they
cannot fully replicate the complex interactions that occur between multiple cell types
in vivo
, both
within an organ and between organs, and cannot be used for true pharmacokinetic and toxicokinetic
studies even though they can examine the potential for particles to cross cell boundaries (Stone
et al., 2009). Moreover, cells in culture do not experience the range of pathogenic effects observed
in the human body. Therefore, unfortunately, the data from cell culture systems could be misleading
and invalid.
