Civil Engineering Reference
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nanomaterials. A simple solution to this liability is to incorporate more than
one cell type (often different cell lineage) and to conduct multiple cytotox-
icity assays simultaneously for a range of reasonable doses and durations
of exposure. Recently, Shaw et al. (2008) demonstrated that the predictive
power of an in vitro assay could be greatly improved by including multiple
cell lines (4 different cell lines) and multiple doses of nanomaterials. It
should be emphasized that the data quality from cell-based HTS assays is
to a strong degree dependent on the equipment and the experimental con-
ditions. Special attention has to be paid to artifacts stemming from the use
of microtiter plates such as edge effects due to the evaporation of culture
media or irregularities arising from liquid handling errors. Hence, any cell-
based assay has to be thoroughly optimized for HTS, validated on the
equipment used for the toxicity screening and it is necessary to standardize
the experimental conditions employing appropriate negative and positive
controls on each microtiter plate. We will now review in detail HTS
approaches toward three of the most important nanotoxicity paradigms:
mutagenicity, cytotoxicity, and oxidative stress.
7.4.3 HTS for mutagenicity, cytotoxicity and oxidative
stress effects of MNMs
The fi rst in vitro toxicology assay was the Ames test, which dates back to
the 1970s and it is still in use today. In this test, several strains of bacteria
- Salmonella typhimurium with various mutations in the histidine produc-
tion pathway - are being exposed to a mutagenic material in question.
Mutations resulting in a frame shift mutation or reversing a point mutation
results in a restoration of the respective histidine production pathway gene
and results in the production of histidine. The readout is growth on histidine
defi cient media. Several commercial HTS versions of this test exist for plate
readers. For example, damaging effects of MNMs on DNA can be detected,
triggering using a reporter gene assay system in which luciferase is put
under the recN promoter which gets triggered by the SOS DNA repair
response. The commercial name of this test is 'VitoTox' test (Verschaeve
et al. , 1999).
Care should be taken when comparing the results from assays: while the
Ames test detects mutagenicity, the VitoTox test detects genotoxicity.
GreenScreen is another assay similar in principle to the VitoTox test. In
GreenScreen (Van Gompel et al. , 2005), the RAD54 promotor drives green
fl uorescent protein (GFP) expression in yeast. RAD54 is involved in DNA
double strand breakage response and the activation of the repair pathway
by a genotoxic MNMs leads to GFP expression. In the commercial product
RadarScreen, the GFP is replaced with
￿ ￿ ￿ ￿ ￿ ￿
-galactosidase. Galactosidase
can be read on a plate reader using various colorigenic or luminogenic
β
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