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

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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  1. 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

  2. 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

  3. 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

  4. (+) 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

  5. 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

  6. 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

  7. 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)

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

  9. 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

  10. 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

  11. 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

  12. 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

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

  14. 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

  15. 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

  16. 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)

  17. 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

  18. 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

  19. 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?

  20. ~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

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

  22. 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?

  23. 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

  24. 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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