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, Mount Vernon Hospital, Northwood, United Kingdom

2. Brief history of the early use of DCFH 2 How the use of DCFH 2 and DHR was introduced into cellular systems for the detection of ROS Recent and current research on the chemistry underling the use of DCFH 2 and DHR in biological systems Practical guidelines to the use of DCFH 2 and DHR in biological systems Overview

3. I. Early use of DCFH 2 DCF Keston and Brandt, 1965 Cathcart, Schwiers and Ames, 1984 O Cl OH Cl H COOH HO 2,7-dichlorodihydrofluorescein DCFH 2 LOOH DCFH 2 + HRP (or hematin) Measurement of hydroperoxides in biological samples (an alternative to the TBA test and iodide assay)

4. DCFH 2 2,7-dichlorodihydrofluorescein non-fluorescent DCF 2,7-dichlorofluorescein fluorescent Peroxide (H 2 O 2 or LOOH) oxidation Importance of catalyst O Cl OH Cl H COO ─ HO O COO ─ Cl HO O Ex 501 nm Em 521 nm (+) CATALYST HRP or haematin (+)

5. DCFH 2 oxidation to DCF involves two single-electron oxidation steps See Rota et all, 1999 OH O Cl H COOH HO DCFH 2 DCF O COOH Cl HO O O COOH Cl HO OH DCFH Compound I or II (  1e ─ ) Compound I or II (  1e ─ ) -2e 

6. Fe 3+ N N Resting enzyme Fe 4+ N N O + Compound I H2O2H2O2 H2OH2O +2e ─ Fe 4+ N N O Compound II AH 2 AH + H +  1e ─ AH 2 AH + OH ─  1e ─ Interaction of peroxidases with H 2 O 2

7. DHR was shown to be three times more sensitive than DCFH 2 in the detection of oxidants produced during the respiratory burst of neutrophils ( Rothe et al.,1988 ) Compound I or II (  1e ─ ) DHR O COOMe Cl H2NH2N NH 2 + Compound I or II (  1e ─ ) DHR NH 2 O Cl H COOMe H2NH2N Dihydrorhodamine 123 (taken up directly by cells) Rhodamine Rh O COOMe Cl H2NH2N NH 2 + -2e 

8. II. Application of DCFH 2 and DHR to the detection of ROS in cellular systems – the forgotten catalyst

9. 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 Cells no Bcl-2 cell death Cells Bcl-2 cells survive 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

10. Modelling mitochondrial O 2 – /H 2 O 2 production using xanthine oxidase M. J. Burkitt and P. Wardman (2001) Biochem. Biophys. Res. Commun. 282, 329-333 O2O2 xanthine oxidase, hypoxanthine O 2 – + H 2 O 2 DCFH 2 DCF time (min) DCF formation (fluorescence intensity) cyt c 0 2 4 6 0.18  M O 2 – min -1 control + cyt c 0510152025 0 2 4 6 control + cyt c 1.66  M O 2 – min -1

11. Bcl-2 – – release into cytosol O2O2 O2–O2– H2O2H2O2 SOD cytochrome c compound I DCFH 2 ++ DCF cyt c H2OH2O GSH GSSG GtPx cyt c cyt c oxidase NADH FADH 2 quinone cycle O2O2 2 H 2 O +4e 

12. DCFH 2 and GSH compete for reaction with cyt c Cyt c–Fe 3+ + H 2 O 2 GSH DCFH 2 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

13. Recent and current research on the chemistry underling the use of DCFH 2 and DHR in biological systems

14. HO Cl O Cl H COOH HO DCFH 2 DCF O CO 2 H ClCl HOO O COOH ClCl HOOH DCFH ee ee ee NAD(P)H, AscH ,GSH NAD , AscH , GS  ee O CO 2 H ClCl HOO 1,3 * hv See Marchesi et al. 1999 O2O2 O2O2 ee H2O2H2O2 ee O2O2 O2O2

15. Determination of the reduction potential of DCF/DCF   (DCFH  ) via equilibration with redox indicators - observed using pulse radiolysis  0.75 V at pH 7.4 pH 46810 -0.8 -0.6 -0.4 NAD AQS MV E  E O 2 /O 2   =  0.33 V

16. 0 40 80 0 20 40 no O 2 3.8% O 2 Decay of the DCF   (DCFH  ) in absence and presence of oxygen observed by pulse radiolysis radical concentration (AU) time (  s) 390 nm

17. pKa = 7.65 ± 0.20 Rate constant for the reduction of oxygen by DCF   (DCFH  ) at various pH values O COOH Cl HOOH O2O2 O2O2 DCFH  O COOH Cl HO O DCF k ~ 10 8 M  1 s  1

18. ee NADH, AscH ,GSH NAD , AscH , GS  HO Cl O Cl H COOH HO DCFH 2 DCF O CO 2 H ClCl HOO O COOH ClCl HOOH DCFH See Rota et al. 1999 ee ll  O CO 2 H CC OO Phenoxyl radical Oxidising radical Reducing radical ee ee ee H2O2H2O2 O2O2 O2O2 ee ee O2O2 O2O2

19. kinetics? H2O2H2O2 O 2 ― (SOD) No reaction with DCFH 2 or DHR + O 2 NO 2 No reaction with DCFH 2 or DHR + Fe 2+ Oxidation of DCFH 2 and DHR OH ONOO ― ONOOCO 2 ― + CO 2 NO NO 2 CO 3 ― + peroxidase Oxidation of DCFH 2 / DHR Compound I/II Interaction of leuco dyes with free radicals

20. OHNO 2 CO 3 ─ DCFH 2 1.3  10 10 1.3  10 7 2.6  10 8 DCF 9.2  10 9 1.7  10 5 2.7  10 8 DHR 1.8  10 10 < 10 5 6.7  10 8 Rh 1.6  10 8 < 10 5 3.6  10 6 GSH 9  10 9 2  10 7 5  10 6 Ascorbate 1  10 9 4  10 7 1  10 9 Urate 7  10 9 2  10 7 Cysteine 2  10 10 5  10 7 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

21. Practical guidelines to the use of DCFH 2 and DHR in biological systems

22. If : O 2 – or H 2 O 2 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 DCFH 2 /DHR without catalyst (e.g. where NOS is uncoupled due to tetrahydrobiopterin oxidation) 1. Try to determine the species responsible for DCFH 2 /DHR oxidation in the experimental system After considering these factors, is increased H 2 O 2 generation the only explanation for an increased in DCF formation?

23. GSH, AscH  and NAD(P)H: will compete with DCFH 2 /DHR Depletion of these will result in greater DCFH 2 /DHR oxidation 2. Consider competition between DCFH 2 /DHR and antioxidants for reaction with oxidants O2O2 H2O2H2O2 cyt c compound I cyt c DCF + DCFH 2 (from mitochondria) GSH GS  AscH ― Asc  ― DCFH 2 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 DCFH 2 from oxidation by RNS NADH NAD 

24. Conclusions DCFH 2 and DHR are useful probes for oxidants in biological systems if accompanied by a ‘health warning’: oxidation is non-specific oxidation by H 2 O 2 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