Page 232 - Environmental Nanotechnology Applications and Impacts of Nanomaterials

Principles and Procedures to Assess Nanomaterial Toxicity 217

enzyme to reduce a tetrazolium salt to water-soluble formazan, which
can be measured by absorption at 492 nm [47]. The major advantage of
these screening assays is the ease with which they can be performed and
the ability to compare a large number of NM simultaneously.
ROS generation in cells can be detected by fluorescent dyes such as
dichlorofluorescein-diacetate (DCFH-DA) and MitoSOX™ Red [41].
DCFH-DA is a nonpolar compound that readily diffuses into cells, where
it is hydrolyzed to the nonfluorescent polar derivative, DCFH, which is
.
trapped in the cell. In the presence of a H O and hydroxyl (OH ) radi-
2
2
cals, DCFH is oxidatively modified into a highly fluorescent derivative,
2,7-dichlorofluorescein (DCF). DCF fluorescence can be detected in cells
by a flow cytometry procedure. MitoSOX Red is a novel fluorogenic indi-

cator offering direct measurement of superoxide (O ) production in live
2
cells. This cell-permeant dye is rapidly and selectively concentrated in

mitochondria, where oxidation by O but not other oxygen or nitrogen
2
species leads to the formation of a fluorescent product that interacts with
nucleic acids. MitoSOX Red fluorescence is also detectable by flow cytom-
etry. Both flow cytometry procedures can be adapted to study the kinet-
ics of ROS generation as well as dose response relationships.
Whether a biological response follows cellular ROS production is
dependent on the magnitude of the response, as well as the antioxidant
buffering capacity [11]. If the antioxidant defense capability of the cell
is overwhelmed, excess ROS production leads to cellular damage as
well as GSH depletion. The GSH content of the cell is an important
determinant of redox equilibrium and if perturbed could elicit further
cellular responses. Cellular thiol levels can be assessed with a thiol-
interactive fluorescent dye, monobromobimane (MBB), using a flow
cytometry procedure [41]. Since GSH makes the biggest contribution
towards the thiol content of the cell, this assay is helpful in predicting
whether NM elicit oxidative stress.
According to the hierarchical oxidative stress hypothesis, cells respond
to even minimal levels of oxidative stress with a protective antioxidant
response [31]. This pathway is dependent on transcriptional activation
of phase II gene promoters by the transcription factor, Nrf2 [27]. Heme
oxygenase 1 (HO-1) is a prime example of a phase II enzyme that medi-
ates antioxidant, anti-inflammatory, and cytoprotective effects, and it
is useful as a marker for particle-induced oxidative stress effects [28].
HO-1 and phase II enzyme expression can be assessed by immuno-
blotting, real-time PCR, or reporter gene assays that reflect the activa-
tion of the antioxidant response element by Nrf2 [27, 28]. The value of
these assays is the sensitivity of the phase II response to oxidative
stress. It is possible to discern phase II enzyme expression at minimal
levels of oxidative stress that are not necessarily related to oxidant
injury. Not only is phase II enzyme expression indicative of the ability