detection of reactive oxygen and nitrogen species using leuco dyes (dcfh 2 and dhr) marta wrona,...
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![Page 1: Detection of reactive oxygen and nitrogen species using leuco dyes (DCFH 2 and DHR) Marta Wrona, Mark Burkitt and Peter Wardman Gray Cancer Institute,](https://reader036.vdocuments.mx/reader036/viewer/2022062515/56649cc15503460f94988e23/html5/thumbnails/1.jpg)
Detection of reactive oxygen and nitrogen species using
leuco dyes (DCFH2 and DHR)
Marta Wrona, Mark Burkitt and Peter WardmanGray Cancer Institute, Mount Vernon Hospital, Northwood,
United Kingdom
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Brief history of the early use of DCFH2
How the use of DCFH2 and DHR was introduced into cellular systems for the detection of ROS
Recent and current research on the chemistry underling the use of DCFH2 and DHR in biological systems
Practical guidelines to the use of DCFH2 and DHR in biological systems
Overview
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I. Early use of DCFH2
DCF
Keston and Brandt, 1965Cathcart, Schwiers and Ames, 1984
O
Cl
OH
ClH
COOH
HO
2,7-dichlorodihydrofluorescein
DCFH2
LOO
H
DCFH2 + HRP (or hematin)
Measurement of hydroperoxides in biological samples (an alternative to the TBA test and iodide assay)
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DCFH2
2,7-dichlorodihydrofluorescein
non-fluorescent
DCF 2,7-dichlorofluorescein
fluorescent
Peroxide (H2O2 or LOOH)
oxidation
Importance of catalyst
O
Cl
OH
ClH
COO─
HO O
COO─
Cl Cl
HO O
Ex 501 nm Em 521 nm
(+) (+)
CATALYSTHRP or haematin(+)
(+)
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DCFH2 oxidation to DCF involves two single-electron oxidation steps
See Rota et all, 1999
OHO
Cl ClH
COOH
HO
DCFH2 DCF
O
COOH
Cl Cl
HO O
O
COOH
Cl Cl
HO OH
•
DCFH•
Compo
und
I or I
I
(1e
─ )
Compo
und
I or I
I
(1e
─ )
-2e
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Fe3+
N
N
Resting enzyme
Fe4+
N
N
O
•+
Compound I
H2O2
H2O+2e─
Fe4+
N
N
O
Compound II
AH2•AH + H+
1e─
AH2
•AH + OH─
1e─
Interaction of peroxidases with H2O2
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DHR was shown to be three times more sensitive than DCFH2 in the detection of oxidants produced during the respiratory burst of neutrophils (Rothe et al.,1988)
Compound I or II
(1e─ )
DHR•
O
COOMeCl Cl
H2N NH2+
•Compound I o
r II
(1e─ )
DHR
NH2O
Cl ClH
COOMe
H2N
Dihydrorhodamine 123(taken up directly by cells)
Rhodamine
Rh
O
COOMe
Cl Cl
H2N NH2+
-2e
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II. Application of DCFH2 and DHR to the detection of ROS in cellular systems – the forgotten catalyst
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Role of ROS in cell death pathways
Concluded that Bcl-2 suppresses the production of common mediator of cell death, i.e. reactive oxygen species – but the role of catalyst was overlooked
Cellsno Bcl-2
cell death
CellsBcl-2
cells survive
GSH depletion
GSH depletion
Kane et al., (1993) Science 262, 1274, Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species
high DCF
low DCF
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Modelling mitochondrial O2•–/H2O2 production using
xanthine oxidase
M. J. Burkitt and P. Wardman (2001) Biochem. Biophys. Res. Commun. 282, 329-333
O2
xanthine oxidase,hypoxanthine
O2•– + H2O2
DCFH2 DCF
time (min)
DC
F fo
rma
tion
(flu
ore
sce
nce
inte
nsity
)
cyt c
0
2
4
6
0.18 M O2•– min-1
control
+ cyt c
0 5 10 15 20 25
0
2
4
6
control
+ cyt c
1.66 M O2•– min-1
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Bcl-2––
releaseinto cytosol
O2
O2•–
H2O2
SOD
cytochrome c compound I
DCFH2
+ +
DCF
cyt c
H2O
GSH
GSSGGtPx
cyt c cyt c oxidase
NADH
FADH2
quinonecycle
O2 2 H2O+4e
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DCFH2 and GSH compete for reaction with cyt c
Cyt c–Fe3+ + H2O2
GSH
DCFH2 DCF
competing reactions
The level of DCF fluorescence is a function of both free [cyt c] and [GSH] / [GSSG]
(Also true for DHR oxidation)
Cyt c compound I
GSSG
See Lawrence et all, (2003) J. Biol. Chem. 278, 29410
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Recent and current research on the chemistry underling the use of DCFH2 and DHR in biological systems
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HO
Cl
O
ClH
COOH
HO
DCFH2
DCF
O
CO2H
Cl Cl
HO O
O
COOH
Cl Cl
HO OH
•
DCFH•
e
e
e
NAD(P)H, AscH,GSH
NAD, AscH, GS
e
O
CO2H
Cl Cl
HO O1,3
*
hv
See Marchesi et al. 1999
O2
O2
e
H2O2
e
O2
O2
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Determination of the reduction potential of DCF/DCF (DCFH) via equilibration with redox indicators - observed using pulse radiolysis
0.75 Vat pH 7.4
pH4 6 8 10
-0.8
-0.6
-0.4
NAD
AQSMV
E
E O2/O2 = 0.33 V
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0
40
80
0 20 40
no O2
3.8% O2
Decay of the DCF (DCFH) in absence and presence of oxygen observed by pulse radiolysis
rad
ica
l co
nce
ntr
ati
on
(A
U)
time (s)
390 nm
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pH
6 7 8 9
10-8
k (
M-1
s-1
)
1.2
8.0
4.0
pKa = 7.65 ± 0.20
Rate constant for the reduction of oxygen by DCF
(DCFH) at various pH valuesO
COOHCl Cl
HO OH
•
O2
O2
DCFH
O
COOH
Cl Cl
HO O
DCF
k ~ 108 M1 s1
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e
NADH, AscH,GSH
NAD, AscH, GS
HO
Cl
O
ClH
COOH
HO
DCFH2
DCF
O
CO2H
Cl Cl
HO O
O
COOH
Cl Cl
HO OH
•
DCFH•
See Rota et al. 1999
e
l l
O
CO2H
C C
O O
Phenoxyl radical Oxidising radical
Reducing radical
e
e
e
H2O2
O2
O2
e
e
O2
O2
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kinetics?
H2O2
O2•―
(SOD)
No reaction with DCFH2 or DHR
No reaction with DCFH2 or DHR
+ O2
NO2•
No reaction with DCFH2 or DHR
+ Fe2+
Oxidation of DCFH2 and DHR
•OH
ONOO―ONOOCO2―
+ CO2
•NO
NO2•CO3
•―+ peroxidase
Oxidation of DCFH2 / DHR
Compound I/II
Interaction of leuco dyes with free radicals
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•OH NO2• CO3
•─
DCFH2 1.3 1010 1.3 107 2.6 108
DCF 9.2 109 1.7 105 2.7 108
DHR 1.8 1010 < 105 6.7 108
Rh 1.6 108 < 105 3.6 106
GSH 9 109 2 107 5 106
Ascorbate 1 109 4 107 1 109
Urate 7 109 2 107
Cysteine 2 1010 5 107 5 107
Rate constants, k (M-1 s-1) ~4% ~67%
Wrona et al. (2004) Free Radical Biol. Med. 38, 262-270
0.3 mM
5 mM
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Practical guidelines to the use of DCFH2 and DHR in biological systems
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If : O2•– or H2O2 involved (e.g. from mitochondria or NADPH
oxidase), Do: 1) consider which haem protein / metal is catalysing oxidation 2) consider how its concentration might change
iron (release from storage proteins during oxidative stress)
cytochrome c (release from mitochondria during apoptosis)
myeloperoxidase (inflammation – macrophages/PMNs)
Peroxynitrite-derived species rapidly oxidize DCFH2/DHR without
catalyst (e.g. where NOS is uncoupled due to tetrahydrobiopterin oxidation)
1. Try to determine the species responsible for DCFH2/DHR oxidation in the experimental system
After considering these factors, is increased H2O2 generation the only explanation for an
increased in DCF formation?
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GSH, AscH and NAD(P)H: will compete with DCFH2/DHR Depletion of these will result in greater DCFH2/DHR oxidation
2. Consider competition between DCFH2/DHR and antioxidants for reaction with oxidants
O2 H2O2 cyt c compound I
cyt c
DCF+ DCFH2
(from mitochondria)
GSH
GS
AscH―
Asc―
DCFH2 loading/retention in cells affects [probe]/[GSH] ratio
GSH may be depleted via drug metabolism Ascorbate can auto-oxidise in cell culture media Urate can protect DCFH2 from oxidation by RNS
NADH
NAD
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Conclusions
DCFH2 and DHR are useful probes for oxidants in biological systems if accompanied by a ‘health warning’:
oxidation is non-specific
oxidation by H2O2 requires a catalyst
antioxidants will compete with probe for oxidants or influence catalytic activity
variations in probe loading, catalyst release or antioxidants will change signal even if ‘ROS’ or ‘RNS’ are constant
photochemical effects may be a factor