Detection of reactive oxygen and nitrogen species using leuco dyes dcfh 2 and dhr
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Detection of reactive oxygen and nitrogen species using leuco dyes (DCFH 2 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 DCFH 2

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Detection of reactive oxygen and nitrogen species using leuco dyes (DCFH 2 and DHR)

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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


O

OH

HO

Cl

Cl

H

COOH

I. Early use of DCFH2

Measurement of hydroperoxides in biological samples (an alternative to the TBA test and iodide assay)

DCFH2 + HRP (or hematin)

DCF

LOOH

Keston and Brandt, 1965

Cathcart, Schwiers and Ames, 1984

2,7-dichlorodihydrofluoresceinDCFH2


(+)

CATALYST

HRP or haematin

(+)

O

O

OH

HO

HO

O

Cl

Cl

Cl

Cl

H

COO─

COO─

Importance of catalyst

Peroxide (H2O2 or LOOH)

(+)

(+)

oxidation

DCFH2

2,7-dichlorodihydrofluorescein

DCF

2,7-dichlorofluorescein

non-fluorescent

fluorescent

Ex 501 nm Em 521 nm


HO

O

OH

Compound I or II

(1e─)

Cl

Cl

H

COOH

HO

OH

O

Compound I or II

(1e─)

Cl

Cl

-2e

DCFH2

COOH

O

HO

DCF

O

Cl

Cl

DCFH•

COOH

DCFH2 oxidation to DCF involves two

single-electron oxidation steps

See Rota et all, 1999


Resting enzyme

•AH + OH─

H2O2

Fe3+

N

+2e─

AH2

H2O

1e─

O

O

•+

Fe4+

Fe4+

N

N

N

N

N

AH2

•AH + H+

1e─

Compound II

Compound I

Interaction of peroxidases with H2O2


H2N

NH2

O

Compound I or II

(1e─)

Cl

Cl

H

COOMe

H2N

NH2+

O

Compound I or II

(1e─)

Dihydrorhodamine 123

(taken up directly by cells)

Cl

Cl

DHR

COOMe

-2e

NH2+

H2N

O

DHR•

Cl

Cl

COOMe

Rh

Rhodamine

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


high DCF

low DCF

Role of ROS in cell death pathways

Cells

no Bcl-2

cell death

GSH depletion

Cells

Bcl-2

cells survive

GSH depletion

  • Concluded that Bcl-2 suppresses the production of common mediator of cell death, i.e. reactive oxygen species – but therole of catalyst was overlooked

Kane et al., (1993) Science 262, 1274, Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species


Modelling mitochondrial O2•–/H2O2 production using xanthine oxidase

0.18 M O2•– min-1

6

O2

+ cyt c

4

xanthine oxidase,

hypoxanthine

2

control

0

DCF formation (fluorescenceintensity)

O2•– + H2O2

1.66 M O2•– min-1

6

+ cytc

cyt c

4

2

control

DCFH2

DCF

0

0

5

10

15

20

25

time (min)

M. J. Burkitt and P. Wardman (2001) Biochem. Biophys. Res. Commun.282, 329-333


O2

O2•–

Bcl-2

SOD

cyt c

cytochrome c

compound I

H2O2

GSH

GtPx

+

+

GSSG

H2O

DCFH2

DCF

FADH2

quinone

cycle

cyt c

cyt c oxidase

release

into cytosol

+4e

NADH

O2

2 H2O


DCFH2 and GSH compete for reaction with cyt c

GSH

GSSG

competing

reactions

Cyt c compound I

Cyt c–Fe3++ H2O2

DCFH2

DCF

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


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


O

O

H

H

O

H2O2

O2

C

l

C

l

H

O2

C

OOH

e

H

O

O

OH

O2

C

l

C

l

e

NAD,AscH, GS

O2

C

OOH

e

hv

NAD(P)H, AscH,GSH

H

H

O

O

O

O

O

O

1,3

*

C

C

l

l

C

C

l

l

C

C

O

O

H

H

2

2

See Marchesi et al. 1999

e

e

DCFH2

e

DCFH•

DCF


Determination of the reduction potential of DCF/DCF(DCFH)via equilibration with redox indicators - observed using pulse radiolysis

-0.4

E 

AQS

MV

-0.6

0.75 V

at pH 7.4

NAD

-0.8

4

6

8

10

pH

E O2/O2 = 0.33 V


3.8% O2

Decay of the DCF (DCFH)in absence and presence of oxygen observed by pulse radiolysis

390 nm

no O2

80

radical concentration (AU)

40

0

0 20 40

time (s)


O

HO

OH

Cl

Cl

COOH

DCFH

O2

k ~ 108 M1 s1

O2

HO

O

O

Cl

Cl

COOH

DCF

Rate constant for the reduction of oxygen by DCF (DCFH)at various pH values

pKa =

7.65 ± 0.20


O

O

H

H

O

C

l

C

l

H

O

O

OH

H

e

C

OOH

O2

C

l

C

l

e

O2

C

OOH

e

H

O

O

O

O2

H2O2

C

l

C

l

C

O

H

e

2

NAD,AscH, GS

O2

e

NADH, AscH,GSH

e

O

O

O

C

C

l

l

Phenoxyl radical

C

O

H

2

Reducing radical

DCFH2

e

DCFH•

DCF

Oxidising radical

See Rota et al. 1999


Interaction of leuco dyes with free radicals

Oxidation of DCFH2 and DHR

Oxidation of DCFH2 / DHR

+ peroxidase

+ Fe2+

CO3•―

•OH

NO2•

Compound I/II

+ CO2

ONOOCO2―

ONOO―

H2O2

No reaction with DCFH2 or DHR

(SOD)

No reaction with DCFH2 or DHR

No reaction with DCFH2 or DHR

•NO

O2•―

+ O2

NO2•

kinetics?


~67%

~4%

0.3 mM

5 mM

Rate constants, k (M-1 s-1)

Wrona et al. (2004) Free Radical Biol. Med. 38, 262-270


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-derivedspecies 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

+ DCFH2

DCF

GS

GSH

O2

H2O2

cyt c compound I

(from mitochondria)

AscH―

NADH

cyt c

Asc―

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


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