Transcript Detection of reactive oxygen and nitrogen species using leuco dyes (DCFH2 and DHR) Marta Wrona, Mark Burkitt and Peter Wardman Gray Cancer Institute, Mount.

Detection of reactive oxygen
and nitrogen species using
leuco dyes (DCFH2 and DHR)
Marta Wrona, Mark Burkitt and Peter Wardman
Gray Cancer Institute, Mount Vernon Hospital, Northwood,
United Kingdom
Overview
 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
I. Early use of DCFH2
Measurement of hydroperoxides in biological samples
(an alternative to the TBA test and iodide assay)
DCFH2 + HRP (or hematin)
O
HO
Cl
H
OH
Cl
COOH
2,7-dichlorodihydrofluorescein
DCFH2
Keston and Brandt, 1965
Cathcart, Schwiers and Ames, 1984
Importance of catalyst
Peroxide (H2O2 or LOOH)
(+)
CATALYST
(+) HRP or haematin
O
HO
OH
HO
O
O
(+) (+)
Cl
Cl
H
COO─
Cl
Cl
oxidation
COO─
DCFH2
DCF
2,7-dichlorodihydrofluorescein
2,7-dichlorofluorescein
non-fluorescent
fluorescent
Ex 501 nm Em 521 nm
DCFH2 oxidation to DCF involves two
single-electron oxidation steps
HO
Cl
O
H
HO
Cl
DCFH2
OH
HO
Cl
Cl
OH
COOH O
•
-2e
Cl
COOH
DCFH•
See Rota et all, 1999
O
O
Cl
COOH
DCF
Interaction of peroxidases with H2O2
Resting enzyme
•AH
+ OH─
AH2 
H2O2
Fe3+
1e─
H2O
N
O
O
N
Fe4+
Fe4+
1e─
N
N
+2e─
•AH
Compound II
+ H+
•+
N
AH2
N
Compound I
H2N
Cl
O
H
DHR
H2N
NH2
Cl
COOMe
H2N
-2e
O
•
Cl
Cl
Cl
COOMe
NH2+
Cl
COOMe
NH2+
O
Rh
Rhodamine
Dihydrorhodamine 123
(taken up directly by cells)
DHR•
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)
II. Application of DCFH2 and DHR
to the detection of ROS in cellular
systems – the forgotten catalyst
Role of ROS in cell death pathways
Cells
no Bcl-2
GSH depletion
cell death
high DCF
Cells
Bcl-2
GSH depletion
cells survive
low DCF
 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
Kane et al., (1993) Science 262, 1274, Bcl-2 inhibition of neural death:
decreased generation of reactive oxygen species
–
O2
xanthine oxidase,
hypoxanthine
O2•– + H2O2
cyt c
DCFH2
DCF
DCF formation (fluorescence intensity)
Modelling mitochondrial O2• /H2O2 production using
xanthine oxidase
0.18 M O2•– min-1
6
+ cyt c
4
2
control
0
1.66 M O2•– min-1
6
+ cyt c
4
2
control
0
0
5
10
15
20
25
time (min)
M. J. Burkitt and P. Wardman (2001) Biochem. Biophys. Res. Commun. 282, 329-333
FADH2
quinone
cycle
cyt c
cyt c oxidase
+4e
release
into cytosol
NADH
O2
O2
–
–
•–
O2
2 H2O
Bcl-2
SOD
H2O2
GSH
cyt c
cytochrome c
compound I
GtPx
+ +
GSSG
H2O
DCFH2
DCF
DCFH2 and GSH compete for reaction with cyt c
GSH
Cyt
c–Fe3+
+ H2O2
Cyt c compound I
GSSG
competing
reactions
DCFH2
The level of DCF fluorescence is a
function of both free [cyt c] and
[GSH] / [GSSG]
(Also true for DHR oxidation)
See Lawrence et all, (2003) J. Biol. Chem. 278, 29410
DCF
Recent and current research on the
chemistry underling the use of DCFH2
and DHR in biological systems
O
HO
Cl
OH
Cl
H
COOH
HO
O
OH
e
e
Cl
•
COOH
DCFH2
DCFH•

O2
O2
e NAD, AscH, GS
NAD(P)H,
O2
Cl
e
O2
HO
O
O
e
Cl
Cl
CO2H
e
hv
AscH,GSH
1,3
HO
H2O2
O
O
Cl
DCF
*
Cl
CO2H
See Marchesi et al. 1999
Determination of the reduction potential of
DCF/DCF (DCFH) via equilibration with redox
indicators - observed using pulse radiolysis
-0.4
E 
0.75 V
at pH 7.4
-0.6
AQS
MV
NAD
-0.8
4
E O2/O2 = 0.33 V
6
8
pH
10
radical concentration (AU)
Decay of the DCF (DCFH) in absence and
presence of oxygen observed by pulse radiolysis
390 nm
no O2
80
40
3.8% O2
0
0
20
time (s)
40
Rate constant for the reduction of oxygen by
DCF (DCFH) at various pH values
-1 -1
10 k (M s )
1.2
pKa =
7.65 ± 0.20
HO
O
Cl
•
DCFH
OH
Cl
COOH
8.0
O2
-8
k ~ 108 M1 s1
O2
4.0
HO
Cl
6
7
8
pH
9
DCF
O
O
Cl
COOH
O
HO
OH
Reducing radical
Cl
Cl
H
COOH
e
e
HO
O
Cl
OH
•
COOH
DCFH2
H2O2
O2
DCFH•

O2
e
O2
HO
e
NADH, AscH,GSH
O
O
Cl
CO2H
e
e
DCF
O
Cl
Phenoxyl radical
O
Cl
e NAD, AscH, GS

O
See Rota et al. 1999
O2
Cl
Cl
CO2H
Oxidising radical
Interaction of leuco dyes with free radicals
Oxidation of DCFH2 and DHR
CO3•― NO2•
ONOOCO2
― + CO2
Oxidation of DCFH2 / DHR
+ Fe2+
•OH
ONOO―
+ peroxidase
H2O2
Compound I/II
No reaction with
DCFH2 or DHR
(SOD)
No reaction with
DCFH2 or DHR
•NO
O2•―
No reaction with
DCFH2 or DHR
+ O2
NO2•
kinetics?
Rate constants, k (M-1 s-1)
~4%
~67%
NO2•
CO3•─
DCFH2 0.3 mM 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
9  109
2  107
5  106
Ascorbate
1  109
4  107
1  109
Urate
7  109
2  107
Cysteine
2  1010
5  107
•OH
GSH
5 mM
Wrona et al. (2004) Free Radical Biol. Med. 38, 262-270
5  107
Practical guidelines to the use of
DCFH2 and DHR in biological systems
1. Try to determine the species responsible for
DCFH2/DHR oxidation in the experimental system
 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)
After considering these factors, is increased
H2O2 generation the only explanation for an
increased in DCF formation?
2. Consider competition between DCFH2/DHR and
antioxidants for reaction with oxidants
GS
O2


GSH
H2O2
(from mitochondria)
cyt c
cyt c compound I
AscH―
Asc
―
+ DCFH2
DCF
NADH
NAD
 GSH, AscH and NAD(P)H:
 will compete with DCFH2/DHR
 Depletion of these will result in greater DCFH2/DHR oxidation




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
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