Biology Reference
In-Depth Information
Cytochrome c
Dihydroethidium (hydroethidine)
The reduction of cytochrome c has been
widely used to estimate specifically ROS
release in neutrophils stimulated with dif-
ferent agents. The reaction is inhibited
almost 100% by SOD addition. The product
of the reaction: cytochrome c [Fe (III)] + O 2 •−
O 2 + cytochrome c [Fe (II)] can be analysed
at 550 nm. Although this assay is even less
sensitive than lucigenin for determining the
extracellular production of O 2 •− , it basically
provides the same information.
This probe is cell permeable and can be oxi-
dized by superoxide to form the ethidium
cation, which has a strong fluorescence.
Because hydroethidine (HE) could be oxi-
dized by other ROS, there is still some
debate as to whether the ethidium cation is
really a specific product for the presence of
O 2 •− . An HPLC analysis of the HE oxidation
products promoted by superoxide revealed
a peak corresponding to a new substance,
which was assigned to 2-hidroxietidium.
The latter has different fluorescence proper-
ties to the ethidium cation and its specific
recognition should be performed by HPLC-
FLD (Zhao et al ., 2003, 2005; Fernandes
et al ., 2007). This compound has a minor
tendency to produce superoxide by redox
recycling and is used for detection of intra-
cellular ROS. Disadvantages are that it has
high photolability and that in cells undergo-
ing apoptosis by the intrinsic pathway the
release of cytochrome c may lead to artefac-
tual oxidation of hydroethidine (Zielonka
et al ., 2008).
Tetrazolium salts: NBT; MTT, XTT, WST-1
All these compounds have in common their
ability to be reduced by reactive species to
form a highly coloured formazan that can be
analysed using spectrophotometry and vis-
ualized in cells. These salts are particularly
sensitive to O 2 •− generating the radical tetra-
zoinil, which dismute to form the water-
insoluble blue formazan. Formazan must be
solubilized for its quantitation and there-
fore attempts have been carried out using
MTT or the WST-1 for the same purpose
because they produce derivatives with
increased water solubility. WST-1 com-
pounds have low cost, less probability of
dismutation of O 2 •− to H 2 O 2 and their reduc-
tion is almost 100% inhibited by SOD, sug-
gesting low cell permeability.
Dihydrorhodamine 123 and
2,7-dichlorodihydrofluorescein
Dihydrorhodamine (DHR) is a lipophilic
probe sensitive to hydrogen peroxide. Once
inside the cell DHR undergoes oxidation
and one of its amino groups tautomerizes to
the imino form (Rho123), preventing it from
leaving the cell (Henderson and Chappell,
1993). 2,7-Dichlorodihydrofluorescein (DCFH)
is a derivative of fluorescein that can be oxi-
dized by ROS generating intense fluorescent
dichlorofluorescein (DCF). The diacetate
derivative of DCFH is apolar and non-
fluorescent and thus can enter cells, where
it can serve as substrates for esterases to
release DCFH. This latter is polar and is
therefore trapped inside the cell. The probes
respond to both ROS and RNS (Crow, 1997).
The main disadvantage of both DHR and
DCFH is high sensitivity to photo-oxidation
and a certain tendency to leak away from
cells in long-term assays. Although both
probes could be used, some authors
preferred DHR instead of DCFH for H 2 O 2
Lucigenin
As indicated earlier, lucigenin can be used
for its higher selectivity and sensitivity to
O 2 •− production compared with tetrazolium
salts. Superoxide is able to reduce luci-
genin to form dioxetane. The latter is
unstable and breaks spontaneously to form
N-methylacridone in an excited state, which
leads to light emission. Because lucigenin is
highly selective to O 2 •− , its detection does
not depend on the presence of MPO as it
does for luminol. Although lucigenin can
promote O 2 •− generation by redox recycling,
the amount produced is minimal. Lucigenin
is not a permeable molecule and therefore
when using SOD, the emission of light is
practically inhibited by 100%.
 
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