Biomedical Engineering Reference
In-Depth Information
detecting the genotoxic effects of NPs, but some concerns have to be taken into account. The Ames
test has also been used, but its suitability is still under debate.
17.5.1 c
oMet
a
ssay
Compared with other genotoxicity assays, the comet assay appears to find positive results for NPs
most consistently, which may be attributed to its high sensitivity toward capturing DNA damages as
triggered by ROS (Collins 2009).
The standard comet assay or single-cell gel electrophoresis is a sensitive method to measure single-
and double-strand breaks and abasic sites (Collins et al. 2008). The use of the bacterial enzymes, FPG
or endonuclease III or the human enzyme, OGG1 allows the detection of oxidized bases, a more rel-
evant DNA lesion (Azqueta et al. 2009). In the revised bibliography, 33% of the
in vitro
studies using
the comet assay include the detection of oxidized bases by the inclusion of at least one of these enzymes.
The comet assay detects reversible DNA lesions, since strand breaks and oxidized bases can be
repaired. The rejoining of single-strand breaks is very simple, but rejoining double-strand breaks
and the repair of oxidized lesions are more complicated and prone to errors.
Although there are some guidelines and protocols in the literature (Tice et al. 2000; Hartmann
et al. 2003; Collins and Azqueta 2012; Vasquez 2012), as yet there is no OECD guideline for the
in vitro
assay. Nevertheless, a draft OECD guideline for the
in vivo
assay has been published.
The comet assay can be applied to virtually any type of eukaryotic cell if a cell suspension can
be obtained. This gives a big advantage in testing the genotoxicity of NPs
in vivo
, as many tissues
can be checked with small modifications to the assays (e.g., for sperm). The protocol of the
in vitro
and
in vivo
comet assays used nowadays in nanogenotoxicity tests does not differ from the protocol
used to test the genotoxicity of chemical compounds.
Pfaller et al. (2010) reported the presence of aggregates in the tails of comets when high concen-
trations of some NPs were tested
in vitro
; washing steps are important to remove residual NPs. If
still present during the process, they can come in contact with naked DNA during the final process-
ing steps of the comet assay and induce artificial damages (Stone et al. 2009).
17.5.2 M
IcroNucleus
t
est
The MN assay is a genotoxicity test for the detection of subnuclear DNA-containing fragments
in the cytoplasm of interphase cells. MNs are distinct from main nuclei, and may originate from
acentric chromosome fragments (i.e., lacking a centromere) or whole chromosomes that are unable
to migrate to the poles during the anaphase stage of cell division. The assay detects the activity of
clastogenic (with potential to break chromosomes) and aneugenic chemicals (with potential for lag-
ging entire chromosomes) in cells that have undergone cell division during or after exposure. The
in vitro
test is described in OECD TG 487 and the
in vivo
test in OECD TG 474.
Several cell lines or human lymphocytes can be used in the
in vitro
version; bone marrow and/or
peripheral blood cells of animals, usually rodents, are used in the
in vivo
version. MN tests can only
be applied to dividing cells. Lesions detected with this assays are not reversible.
The
in vitro
MN assay is performed using different approaches (Doak et al. 2009; Gonzalez et al.
2011). Some of them use cytochalasin B to inhibit cytokinesis and generate binucleated cells, and it
is in these cells that the MN frequency is scored, increasing the likelihood that the MNs have been
formed during or since treatment rather than as preexisting ones. This compound inhibits endocyto-
sis, an important cell uptake mechanism, which can interfere with the evaluation of the genotoxicity
of some NPs (Doak et al. 2009; Gonzalez et al. 2011); it is important to treat the cells with NPs but
without cytochalasin B during some time.
Washing steps after
in vitro
treatments are also important since the presence of NPs in the prepa-
rations being scored has been reported by Pfaller et al. (2010) when high concentrations of NPs are
used.
