Page 233 - Environmental Nanotechnology Applications and Impacts of Nanomaterials

218 Principles and Methods

of the cell to defend against oxidative stress, but it also acts as a bio-
marker that reflects subtle levels of oxidative stress that might other-
wise be overlooked. Thus, the presence of phase II responses could alert
one to the possibility that the material elicits more harmful oxidative
stress effects, when, for instance, a higher material dose is delivered or
when exposure takes place in a cell type that lacks the ability to syn-
thesize its own GSH, such as neuronal cells.
When antioxidant defenses fail to restore redox equilibrium, escalation
of the level of oxidative stress can lead to cellular injury [11, 33]. One
mechanism of injury is the activation of pro-inflammatory cascades
[11, 33]. The Jun kinase (JNK) and NF- B cascades are redox-sensitive
signaling cascades that are capable of inducing the expression of pro-
inflammatory cytokines and chemokines. The activation of these cas-
cades can be detected by a variety of techniques, including I B
immunoblotting, phosphopeptide immunoblotting, flow cytometry assays,
and electrophoretic mobility shift assays (EMSA) [32]. The expression of
pro-inflammatory cytokines (e.g., TNF-α, IL-8, IL-6, and GM-CSF) can be
detected by enzyme-linked sorbent assays or real-time PCR. Adhesion
molecule expression (e.g., VCAM-1 and ICAM-1) can be discerned by flow
cytometry as well as real-time PCR. The utility of these assays is the ease
with which they can be performed on a number of samples. It is also pos-
sible to combine the individual pro-inflammatory response markers into
multiplex assays that are capable of measuring several cytokines or
chemokines simultaneously. Many of the same inflammation markers
play a role in the pathogenesis of PM-induced disease in vivo and are also
detectable in body fluids and tissues, e.g., bronchoalveolar lavage fluid.
High levels of oxidative stress can induce changes in the intracellu-
2+ 2+
lar free calcium concentration, [Ca ] , or may perturb Ca compart-
i
mentalization in the cell, with the potential to induce toxicity [48]. The
endoplasmic reticulum and mitochondria play important roles in buffer-
2+
ing sudden increases in [Ca ] . ROS participate in thisincrease through
i
2+
inhibition of the sarco/endoplasmic reticulum Ca ATPase (SERCA) or
by inducing inositol 1,3,5-trisphosphate release [48, 49]. This, in turn,
2+ 2+
can lead to increased mitochondrial Ca levels, [Ca ] . Saturation of
m
2+
the mitochondrial Ca buffering capacity triggers further mitochondr-
ial responses that can produce additional ROS production and more
cellular damage. The mitochondrial permeability transition pore (PTP)
2+
is a Ca -regulated channel that can be triggered by a persistent increase
2+
in [Ca ] [50]. Large-scale PTP opening leads to cytochrome c release
m
2+
2+
and apoptosis [50]. [Ca ] and [Ca ] levels can be followed by using
i
m
fluorescent dyes such as Fluo-3 or Rhod-2 and flow cytometry [41].
These assays can be performed on several samples simultaneously.
Their biological significance is the elucidation of cellular responses that
result in cell death.