Biomedical Engineering Reference
In-Depth Information
17.5.3 B
acterIal
r
everse
M
utatIoN
t
est
: a
Mes
t
est
The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity
and, in particular, for point mutation-inducing activity. The principle and the description of the test
method are in OECD TG 471.
The bacterial reverse mutation test uses amino acid-requiring strains of
Salmonella typhimurium
and
Escherichia coli
to detect point mutations, which involve the substitution, addition, or deletion
of one or a few DNA base pairs. The principle of this test is that it detects reversions, or back-muta-
tions, of a mutated gene present in the test strains, restoring the functional capability of the bacteria
to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in
the absence of the amino acid required by the parent test strain.
Surprisingly, test results have been negative whereas
in vitro
mammalian cell tests often give
positive genotoxic responses (Doak et al. 2012; Jomini et al. 2012; Nam et al. 2013). The most likely
hypothesis is that NPs cannot penetrate the walls of bacterial cells, so the Ames test is not suitable
for testing the genotoxicity of NPs (Landsiedel et al. 2009; Singh et al. 2009; Nabeshi et al. 2011;
Woodruff et al. 2012). However, some studies have shown that NPs can cross the bacterial wall of
E. coli
(Brayner et al. 2006) and
Pseudomonas aeruginosa
(Xu et al. 2004).
Nevertheless, Jomini et al. (2012) showed that the medium used during the exposure of bacterial
cells to NPs prevents electrostatic interactions between them, leading to false-negative responses.
They showed that the exposure of bacteria to TiO
2
NPs in a low ionic strength solution at a pH below
the NP isoelectric point makes the assay suitable. More investigations should be done in order to
validate this last hypothesis.
17.5.4 p
revIous
I
n
V
Itro
c
ytotoxIcIty
s
tudIes
A preliminary cytotoxicity test must always be carried out in order to choose the adequate concen-
tration range for
in vitro
genotoxicity studies.
NPs are optically active due to their high absorption and scattering effects, so standard assays
for testing cytotoxicity may not be suitable. In the revised publications, close to 87% of the studies
used these types of assays while close to 13% used the direct counting of cells or a proliferation
assay. The last option is more complete since it conflates cell killing, growth, and cytostatic effects.
The issue of appropriate concentrations for testing the genotoxicity of chemicals is currently
under discussion. Nowadays, concentrations causing 50% or less cytotoxicity are recommended;
chemicals that exert cytotoxic effects are likely to give false-positive results in genotoxicity tests,
the DNA damage being secondary to cytotoxicity.
17.6 TEST CONDITIONS THAT INFLUENCE THE GENOTOXICITY OF NPs
The differences in the responses between studies may be due to the test being used and to the dif-
ferent endpoints being measured, but also to the different test conditions employed.
The dosing obviously influences the genotoxic potential of NPs, but a dose-dependent relationship
is not always clear. Several studies showed an inverse dose-dependent relationship between dose and
cell genotoxicity using different NPs (Ghosh et al. 2010; Sergent et al. 2012). Calarco et al. (2013)
showed that polyethylenimine-NPs uptake by cells was higher at lower concentrations, which indi-
cates a saturation and limited cellular uptake capability when a higher concentration is used. The
aggregation of NPs at higher doses can explain the inverse dose-dependent relationship between the
dose and genotoxicity. Tang et al. (2010) concluded that the instillation of TiO
2
NPs in rats at low doses
could reversibly affect metabolic function because NPs can migrate from the lungs to the liver and
kidneys, but at higher doses, the particles will aggregate and deposit in the lungs without migration.
The movement of NPs through cells may probably be slower than chemical diffusion, so longer
periods of treatment may be adequate; nowadays, there are very few
in vitro
studies with a treatment
