Safety issues relating to nanomaterials for construction applications - Nanotechnology in Eco-efficient Construction - page 148

the peptide part is cleaved off the aminoluciferin hence making it convert-
ible by fi refl y luciferase. While Capase 3/7 would indicate mitochondrial
impairment, Caspase 8/9 activity might point more at infl ammation and
activation of TNF secretion.

Injury responses due to oxidative stress from MNM exposure have been
documented extensively in the literature and oxidative stress is one of the
best-understood mechanisms of MNM mediated toxicity (Oberdorster
et al. , 2005; Nel et al. , 2006, 2009). MNM induced oxidative stress can be
divided into three tiers: antioxidant defense, pro-infl ammatory effects and
cytotoxicity. MNM toxicity is typically mediated by ROS generation which
can be extra- or intracellular and in both cases depletes the gluthathion
redox-equilibrium of the living cell. Each of these response tiers is
initiated by specifi c biological sensors and activation mechanisms. In Tier
1, the transcription factor Nrf2 is activated to enhance the expression of
phase II enzymes, which attempts to restore redox equilibrium. If the level
of oxidant injury cannot be recovered by phase II enzymes, Tier 2 response
commences, signaling pathways such as the mitogen-activated protein
kinase (MAPK), and nuclear factor kappa B (NF-

α and interleukin-1 β

B) cascades are acti-
vated and cells express proinfl ammatory cytokines. These infl ammatory
effects contribute to disease processes such as asthma and atherosclerosis.
At the highest level of oxidative stress (Tier 3), the mitochondrial integ-
rity is compromised and a resulting drop in ATP synthesis and release of
calcium and other pro-apoptotic factors ultimately leads to cell death. Cyto-
toxicity (Tier 3 response) is the most extreme response to particle effects
involving ROS production. Modes of action can involve the shedding of
toxic metal ions that trigger intracellular ROS generation or cationic
nanoparticles or dissolved transition metal interference with the mitochon-
drial electron transduction. As is evident, it is necessary to select multiple
readouts to capture cellular events specifi c to Tiers 1-3.
Oxidative stress can be detected very easily using HCS. For example, a
cocktail of Hoechst 33342 and the mitochondrial membrane potential dye
JC1 detects Tier 2 responses based on mitochondrial membrane depolariza-
tion. A dye cocktail of Hoechst 33342, fl uo-4 (detecting cytosolic Ca 2+ ) and
propidium iodide (detecting membrane damage) is very useful for the
detection of Tier 3 responses in which the mitochondrial membrane loses
integrity, calcium enters the cytosol and the cell membrane disintegrates. In
the oxidative stress paradigm, in vitro and in vivo are correlated extremely
well: for example, ZnO nanoparticles elicit responses in vitro that are analo-
gous to a disease called 'metal fume fever' (Duffi n et al. , 2007). Metal fume
fever is an acute infl ammatory condition of the lung in welders who are
exposed to aerosolized metal oxide nanoparticles. In the workers, one fi nds
the expression of Tier 2-like responses in macrophages and epithelial cells
as demonstrated by the detection of secreted interleukin 8 (IL-8) and

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