Sunrise Free Radical School Society for Free Radical

Sunrise Free Radical School Society for Free Radical Biology and Medicine Washington, DC November 2007 How to Measure ROS and RNS in Biology Some generality and a singlet molecular oxygen case Paolo Di Mascio Departamento de Bioquímica Instituto de Química Universidade de São Paulo, Brasil

Reactive species have , in generally, a very short half-life, are produced in low amounts. Multiple methods of measurement are available today, each with their own benefits and limits. Reactive Species measurement methods must be: -Very, very sensitive -Highly selective -Fast -In situ

How to Measure Reactive Species? Direct Electron paramagnetic resonance, EPR (free radical detection) +Trapping (Spin-traps, “chemical”) Indirect (but specific) Mass spectrometry, MS/MS (ESI, MALDI) +Scavengers Probes (spectroscopic investigation) Luminescence (direct and indirect) Combination of Different Techniques ! Free Radicals in Biology and Medicine B. Halliwell ans J. M. C. Gutteridge, 2007.

Electron Paramagnetic Resonance (EPR) We can detect & measure free radicals and paramagnetic species • “High” sensitivity (nanomolar concentrations) Direct detection e. g. : semiquinones, nitroxides, thiyl, ROO • … Indirect detection Spin-trapping Species: superoxide, hydroxyl, alkyl, NO Spin-traps: DMPO, PBN, DEPMPO, Fe-DTCs We can use EPR to measure free radicals from biological systems (in vivo or ex vivo) Intact tissues, organs … can be measured.

Other Trapping methods Hydroxyl radical OH▪ [Trapping methods (without EPR)] -Reaction with aromatic compounds (Phenylalanine ‘Tyrosine’, Salicylate, … -Attack of OH▪ on 2 -deoxyribose produces a range of products…malondialdehyde Superoxide O 2 ▪- [Fluorescent probes] Dihydroethidium (DHE) conversion to 2 -hydroxyethidium and lucigenin = - Ability to reduce cytochrome c or nitroblue tetrazolium - Superoxide electrodes - Histochemical detection: Conversion of diaminobenzidine (DAB) to an insoluble product Peroxynitrite and other nitrogen species …. Nitric oxide NO ▪ -NO ▪ : -Light emission in the presence of O 3 (excited NO 2▪) -NO electrodes (porphyrinic sensors) -Haemoglobin trapping ( A is measured) -Spin trapping (Haemoglobin, other haem proteins, …) -4, 5 -Diaminofluorescein diacetate (DAF-2 -diacetate), fluorescent product Diaminoanthroquinone, red fluorescent product -“indirect methods”: NO 2 - measurement, use NOS inhibitors, …

Nitration assays ONOO- : ONOO- nitrates many aromatic compounds: tyrosine (3 nitrotyrosine), tryptophan, phenylalanine Reactive halogen species HOCl / HOBr - [taurine assay] Conversion of taurine to a chloramine -bromo- and chlorotyrosine ? Hydrogen peroxide H 2 O 2 - [fluorescent products] Amplex red and DCF-DA - [non fluorescent products] Fluorescent compound scopoletin -Inactivation of catalase by aminotriazole…. in cells… Singlet oxygen 1 O 2……. . O 2 (1 g) in biological system -Monomol and dimol Light emission -Use of scavengers (azide), traps (histidine, anthracene derivatives) and D 2 O effect Studies using low-level / ultraweak / dark chemiluminescence ….

Many methods are available to identify Reactive Species in cultured cells 1 - Cell culture process itself causes oxidative stress 2 -Trypsinization increases ROS 3 - Artifacts are produced -Dichlorofluorescein diacetate (DCF-DA) is deacetylated by esterases to dichlorofluorescein (DCFH) which can be visualized by fluorescence at 525 nm More specific for H 2 O 2? -Dihydrorhodamine 123 (DHR) is used to detect several reactive species Conversion to rhodamine 123, highly fluorescent 536 nm -Luminol and lucigenin “Biomarkers” Of oxidative DNA damage Of lipid peroxidation Of protein damage by reactive species

• • • “Biomarkers” Single and double strand breaks (Comet assay) Oxidative modification of DNA-bases (e. g. 8 -oxo-d. Guo… LC/MS/MS)Of oxidative DNA damage Formation of DNA adducts with oxidized lipids or proteins (e. g. d. Guo-MDA, d. T-Tyr…. LC/MS/MS). O Guan(os)ine H 2 N O N • HN N H 2 N Oxidation Ring opening N OH H DNA base oxidation: oxidation of guanine by OH • N HN N OH • G 8 OH • N O R N H 2 N Reduction R H 2 N N OH H R O O H N HN N • HN O N HN OH N FAPy-Guanine NH R H 2 N N 8 -oxo-Guanine N R

Arachidonic acid oxidation products: “Biomarkers” Of lipid peroxidation Isoprostanes: Relatively stable endproducts that are currently viewed as most reliable marker of lipid oxidation in vivo Isoprostanes comprise four regioisomers with eight stereoisomers. in vivo (Lawson et al. , J. Biol. Chem. 1999: 274, 2444 -24444).

“Biomarkers” Of lipid peroxidation Metabolic Fate of 15 -F 2 t-Iso. P (8 -Iso-PGF 2 ) in Humans OH COOH OH OH 15 -F 2 t-Iso. P -oxidation 5 -reduction OH COOH Major urinary metabolite OH OH A GC/MS assay for F 2 Iso. P-M has been developed 2, 3 -Dinor-5, 6 -Dihydro-15 -F 2 t-Iso. P (F 2 -Iso. P-M) (Roberts LJ, II, et. al. J. Biol. Chem

“Biomarkers” Stable oxidation markers: Tyrosine oxidation, chlorination, and nitration Of protein damage NH 3+ COO– NH 3+ OH COO– – COO Ox Tyr • OH OH Ox Tyrosine NH 3+ OH Tyrosyl radical • O HOCl, Cl 2 COO– NH 3+ NO 2 • NH 3+ o, o’-dityrosine NH 3+ COO– OH Cl NO 2 3, 4, -di. OH-Phe (DOPA) OH 3 -Cl-tyrosine (RHS) OH 3 -NO 2 -tyrosine (RNS)

H DNA / Protein Adducts O R a, b-unsaturated carbonyls (e. g. acrolein, HNE, cyclopentenone prostaglandins) O H N O deoxy. Guanosine H 2 N HO N H OH N N d. R Acr-d. Guo 1&2 N H Acr-d. G 3 Cro-d. G HNE-d. G R O N N R’ H N O R d. R Acrolein Crotonaldehyde Hydroxynonenal (HNE) O O N N d. R Cysteine O H N Lysine S N N N Histidine H N N HN H -R’ -H -CH 3 -CH(OH)-CH 2 -CH 3 HN O R O

Half-life of some reactive species Reactive species Hydroxyl radical ( OH) Alcoxyl radical (RO ) Singlet oxygen (1 O 2) Peroxynitrite anion (ONOO-) Peroxyl radical (ROO ) Nitric oxide ( NO) Semiquinone radical Hydrogen peroxide (H 2 O 2) Superoxide anion (O 2 -) Hypochlorous acid (HOCl) Half-life (s) Physiol conc. (mol/l) 10 -9 10 -6 10 -5 0. 05 – 1. 0 7 1 - 10 minutes/hours spontan. hours/days 10 -9 - 10 -7 spontan. hours/days 10 -12 - 10 -11 10 -9 (accelerated by enzymes) (by SOD accel. to 10 -6) dep. on substrate

Free Radical Reaction Rate

ROS generated by physical or chemical processes can damage biomolecules. -difficulties and how to measure ROS with the singlet oxygen case Photosensitization Enzyme Myeloperoxidase 1 O 2 - e- Ionizing Radiation Oxidative Metabolism UV Laser Pulses Xenobiotics • OH H 2 O 2 • - ROOH DNA Diseases Lethality Proteins Lipids Mutagenesis Carcinogenesis Aging

Singlet Molecular Oxygen in biological systems as an example What’s the Problem? ? . ROO X = HOCl, ONOOH, …. ROOH ? O 2 • ¯ • HO H 2 O 2 1 O ? 2

Singlet Molecular Oxygen in biological systems as an example Direct Luminescence (Light emission) +Quenchers (N 3 -), Solvent effect (Deuterated solvent, D 2 O) Indirect but specific Isotopically labeled oxygen Chemical trapping and HPLC-mass spectrometry (18 O-labeled compounds) EPR Synthesis / Characterization (LAOOH, PCOOH) 18 O-Labeled Compounds Chemical source of 18[1 O 2] -Development of a pure source of isotopically labeled singlet oxygen, 18[1 O ] 2 Tool for mechanistic studies !

Photosensitized generation of singlet O 2

Porphyria Patient Porphyria is a diseases in which pigments called porphyrins accumulate in the skin, bones and teeth. Porphyrin Nucleus Angew-Chem. Int. Ed. , 21, 343 , 1982

Activated leukocytes Bacterial killing: Respiratory burst Fagocytosis Steinbeck et al. (1992) J. Biol. Chem. , 267, 13425 Wentworth et al. (2002) Science, 298, 2195. Babior et al. (2003) Proc. Natl. Acad. Sci. USA, 100, 3031.

Lipid peroxidation Russell Mechanism? Is singlet oxygen generated from lipid hydroperoxides? ONOO-, LH L LOOH LOO 1 O 2 ?

Generation of Singlet Oxygen by the “Russell Mechanism” 1957 Russell proposed that termination reaction of 2 peroxyl radicals involves a tetraoxide intermediary state. Russel J. Am. Chem. Soc. , 79, 3871, 1957 1968 Howard & Ingold showed the formation of singlet oxygen in the reaction　of sec-butylhydroperoxide with Ce 4+. Howard & Ingold J. Am. Chem. Soc. , 90, 1057, 1968 C O 2 R 1 C R 2 O O • H ! (22 kcal/mol) O O C O H O C H (100 kcal/mol) LOO • + LOO • + CH OH + 1 O 2 LOOOOL or 3 C O* + CH OH + O 2 (75 -80 kcal/mol) L=O L-OH O 2

Experimental Strategy I- Synthesis of LA 18 OH Incubation: LA 18 OH + Metal ion (Cerium, Iron, peroxynitrite) LA 18 O → 18 O - 18 O + LA 18 O IV- 18 O LA 18 O [ LA 18 O 18 OLA ] Chemiluminescence 18 1 [ O 2] III II Chemical Trapping of [ x. O(1 g)] with DPA EPR R R + x. O 2(1 g) Ox Ox R R – 6 H 5 R: –C DPA X : : 18 O or 16 O atoms DPAx. O

Part I Synthesis of 18 O-Labelled Linoleic Acid Hydroperoxide (LA 18 OH) Structures of the 4 LAOOH isomers generated from linoleic acid photooxidation under 18 O 2 atmosphere 13 12 10 Linoleic Acid (LA) 9 18 O 2 Methylene Blue Irradiation 9 -LA 18 OH 10 -LA 18 OH 13 -LA 18 OH 12 -LA 18 OH

Synthesis of Phosphatidylcholine Hydroperoxides by Photooxidation using Methylene Blue as a Photosensitizer 1 O 2 OOH Phosphatidylcholine (PC) Phosphatidylcholine Hydroperoxides (PCOOH)

Electrospray ionization mass spectra of LAOOH and LA 18 OH obtained in the negative ion mode. LA 16 OH (M = 312) +4 LA 18 OH (M = 316)

Generation of [18(1 O 2)] from LA 18 O 18 OH / Metal ion (Cerium, Iron) or (peroxynitrite) → LA 18 O R 1 R 2 C H 18 O O 18 2 H LA 18 O 18 R 2 C O 18 18 O O (1 g) 18 O R 1 LA 18 O 18 OLA [ O 18 O] 1 18 ?

Part II Detection and quantification of 1 O 2 by chemical trapping with 9, 10 -diphenylanthrancene (DPA) Mass spectrometry HPLC/MS-MS (18 O-labeled compounds)

A + 1 O 2 A O 2 “ene” type reaction Cycloaddition of Singlet Oxygen Cycloaddition [2+2] Cycloaddition [4+2]

Singlet Molecular Oxygen Quenching Chemical Quenching Q + O 2( 1 g) QO 2 Physical Quenching Q + O 2( 1 g) Lycopene Q + O 2(3 S -g) + heat

Chemical Trapping of [ x. O(1 g)] with DPA - 18 O +. LA 18 O 18 1 [ O 2] . LA 18 O [ LA 18 O 18 OLA ] Chemical Trapping of [ x. O(1 g)] with DPA

ROOH ROS / RNS “Conversion” ! ? Ce 4+, Fe 2+ + ONOOHOCl Russell 1 O PC-OOH LAOOH 2

Chemical Trapping of [ x. O(1 g)] by EAS and Detection of EASx. O by HPLC-ESI-MS Probe EAS x= 16 or 18 EASx. O 2 EAS 16 O LA 16 OH + HOCl m/z LA 18 OH + HOCl EAS 18 O EAS 16 O m/z

Part III Luminescence Light Emission (spectroscopic investigation) Dimol emission O 2(1 g) + O 2(1 g) 2 O 2(3 S-g) + hn (l= 634 and 703 nm) Monomol emission O 2(1 g) O 2(3 S -g) + hn (l= 1274 nm)

Detection and Characterization of 1 O 2 Improvement / “new” systems Singlet Oxygen Monomol light Emission O 2(1 g) O 2(3 -g) + hn ( = 1270 nm) O 2(1 g) Sample Chamber Monochromator Ge-Diode 1 O 2 hn Photomultiplier O 2(3 g-)

Emission spectrum of the hydrogen peroxide-hypochlorite reaction H 2 O 2 + OCl- HOCl + HO 2 - HOOCl + HO- H 2 O + Cl. OO- 1 O 2 + Cl---------------------H 2 O 2 + OCl- 1 O 2 + Cl- + H 2 O Dimol emission O 2(1 g) + O 2(1 g) 2 O 2(3 g) + hn ( = 634 and 703 nm) Khan and Kasha, 1963

Monomol emission O 2(1 g) O 2(3 S -g) + hn (l= 1274 nm) Near-IR

Characterization of singlet oxygen generated in the reaction of LAOOH and Ceric ion by chemiluminescence Dimol light emission at > 570 nm O 2(1 g) + O 2(1 g) 2 O 2(3 S-g) + hn (l= 634 and 703 nm) Monomol light emission at =1270 nm O 2(1 g) O 2(3 S -g) + hn (l= 1270 nm)

The presence of a hydrogen- at the carbon to which the hydroperoxide is attached is essential for the generation of O 2 (1 g). ROOH / HOCl Emissão de luz em 1270 nm (102 counts) O 2 (1 g) 160 O 2 (3 g-) + hn ( =1270 nm) A 140 6 H 2 O 2 120 100 t-Bu. OOH H 3 C C O OH H 3 C 4 80 100 % 60 40 3 2 HOCl 20 0 6 30 60 B 5 90 HO O 4 120 0 150 O OH 30 60 D 5 90 4 H 3 C 2 1 120 150 Cu. OOH 3 14 % HOCl 0 6 LAOOH 3 2 HOCl 1 0 0 C 5 O OH C H 3 C HOCl 1 0 30 60 90 120 150 0 0 Tempo (s) 30 60 90 120 150

EPR spectrum of linoleate peroxyl radicals, LAOO. Reaction of LAOOH with HOCl Part IV Direct detection

Take-Home Message Conclusion: Combination of Different Techniques ! Source For example, lipids? 18 O-labeled DHPN 18 O 2 source Near-IR 2. 4 18 + 18 2 LA 18 O II- Chemiluminescence [ LA 18 O 18 OLA ] Light Emission ( 10 3 cps) I- Synthesis of V- Clean chemical source of 18 O-labeled reacive species, [1 O 2] Clean source O O 18[1 O (1 g) 2. 1 (0, 0) 1 g 3 g 1270 and Visible 1. 8 1. 5 1. 2 0. 9 0. 6 1200 1250 1300 1350 l (nm) HPLC-MS/MS trapping 2] EPR III- Mass Spectrometry IV- EPR

Chemical source R 18 O of [18 O] isotopically labeled singlet oxygen 18[1 O R 18 O 18 1 [O ] 2 + R R ? . . to elucidate mechanism of 1 O 2 reaction towards biological target Excellent tool (+MS) for mechanistic studies of the reaction of singlet molecular oxygen in biological media. 2]

Positive APCI-mass spectra in the MS/MS mode of the of 9, 10 -diphenylanthracene (DPAx. O, x: 16 O or 18 O) DPA 16 O DPA 18 O 25 m. M LA 18 OH + 25 m. M Ce 4+ + 60 m. M DPA/37 C, 1 hour endoperoxides Probe

Energy transfer between singlet (1 g) and triplet (3 g-) molecular oxygen in aqueous solution. “propagation” !