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

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

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)

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(+)

(+)

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

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

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

II. Application of DCFH2 and DHR to the detection of ROS in cellular systems – the forgotten catalyst

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

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

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

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

Recent and current research on the chemistry underling the use of DCFH2 and DHR in biological systems

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

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

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

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

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

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

•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

Practical guidelines to the use of DCFH2 and DHR in biological systems

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?

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

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