Civil Engineering Reference
In-Depth Information
substrates. Going up in the evolutionary chain, the GreenScreen HC assay
relies on a TK6 lymphoblastoma cell line with a growth arrest and damage
gene promoter GADD45
in driving the luciferase reporter.
It is interesting to note that drugs, which are normally tested in these
assays, are exposed to liver enzymes that metabolize the compound in ques-
tion before their use in these assays. The reasoning is that frequently the
metabolites are more toxic than the parent compound. At this point it is
frequently unclear where and how MNMs are processed or broken down
in an organism and it is unclear if exposure to liver enzyme will be useful
for MNM toxicity testing. While results from assays for mutagenicity testing
are quite universally applicable since the carrier of genetic information,
DNA, is universal, this is not the case for cytotoxicity. Cytotoxicity is very
dependent on the presence of a target mediating the cytotoxic effect. Hence
precise knowledge of the mode of action (MOA) is necessary to fully under-
stand and appreciate the cytotoxic potential of any given MNM.
For drugs, dyes allowing for measuring DNA content such as Hoechst
33342 are frequently used for the detection of, e.g., pro-mitotic effects using
HCS approaches. Cell cycle analysis using various dye combinations can be
very useful as well for the detection of hazardous MNMs and are best
executed using HCS. Membrane integrity can be assessed using propidium
iodide (PI) and/or Calcein-AM dyes. While the nucleic acid dye PI does
not pass intact membranes, Calcein AM is a conjugated membrane perme-
able, fl uorogenic fl uoresceine derivative that once in the cell and processed
by esterases can no longer leave the cell - except if the membrane integrity
is compromised. This assay can be performed in a plate reader or HCS
system.
Cytotoxicity of an MNM can show itself as well in the collapse of the
gluthathion level of a cell or radical oxygen species (ROS) formation, which
can be detected in the presence or absence of cells. Gluthathione depletion
can be easily assessed using monochloimane and ROS formation can be
easily measured using dichlorofl uoresceine. Both of these dyes can be used
in a plate reader format. The redox potential of a cell - i.e. gluthathion level
- can be followed using dyes such as MTT or Alamar Blue (Nakayama
et al.
, 1997). While these dyes are relatively cheap, the fl uorescent character
of the nanomaterials might interfere with their detection. Hence the use of
CytoLite (measures NADH) (Chan
et al.
, 2001), ATP-Lite or CellTiter Glo
(measure ATP) might be preferable (Hannah
et al.
, 2001). A combination
of these three assays should enable a fi rst picture of the MOA of the toxicity
of any given nanomaterial.
An interesting twist on apoptosis detection, which is a frequent liability
of toxic MNMs, is the use of Z-DEVD-aminoluciferin for Caspase 3/7 and
LETD-aminoluciferin for Caspase 8/9 (O'Brien
et al.
, 2005). Both sub-
strates have in common that upon activation of the Caspases in question,
α
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