Civil Engineering Reference
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media. A methodic approach is imperative when performing nanotoxicol-
ogy in any assay system to exclude false results. Therefore, it becomes
urgent to develop standard protocols for evaluating nanomaterial stability
during
in vitro
or
in vivo
nanotoxicity studies.
We will now review existing approaches for small molecule HTS toxicol-
ogy screening as many of these approaches and workfl ows currently used
in drug discovery can be potentially adapted towards ENM toxicology
screening.
7.4.2 General considerations for MNM toxicity profi ling
using HTS
HTS approaches for toxicology typically utilize microtiter plates of the 96
or 384 well plate format. These plate formats are formalized by the Society
of Biomolecular Screening and all existing equipment is compatible with
the plate formats.
The screening approaches themselves are either plate reader or high
content screening (HCS) based. Plate reader based assays rely on one of
the classic standard readouts such as luminescence, fl uorescence, absorp-
tion, time-resolved fl uorescence and fl uorescence polarization and measure
the whole well at once. TR-FRET and fl uorescence polarization (FP) are
readouts that are less commonly used for toxicity screening as their cost is
higher than the other three assay readouts mentioned and especially FP is
very sensitive to interference from contaminants and temperature varia-
tions. While plate reader based assays typically have a single parameter,
which is measured at a point in time, HCS offers a complementary approach,
which can be understood as automated microscopy coupled with image
analysis software. It enables the measurement of multiple parameters with
cellular resolution in the same well at the same time and offers a more
detailed view. Moreover, HCS enables the detection of rare cellular events,
segregation of sub-populations of cells from a single well and has the poten-
tial to be more sensitive and also pick up sub-lethal effects. Another impor-
tant factor is that HCS enables phenotypic screening, which can be based
on, e.g., cell morphology or translocation event of a given protein.
HCS-based toxicity screening has signifi cant advantages over classical
plate reader based assays. In a 2006 study (O'Brien
et al.
, 2006), conven-
tional single plate reader based readouts were compared to a fi ve parameter
HCS assay for toxicology prediction power on a set of reference com-
pounds: the single parameter readouts had a very limited sensitivity (<25%)
but high specifi city (about 90%), but HCS had a sensitivity of 93% and a
specifi city of 98%. What is more interesting is the fact that although hepa-
tocyte HepG2 cells were used, 92% of the toxic drugs, which had other
organ toxicities, but no hepatotoxic properties, were detected.
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