Methods for Assessing Oxidative/N itrosative Stress Lukas Kubala, PhD Institute of Biophysics, Brno Email: kubalal@ibp.edu Methodological Approaches Direct Determinations • Low-level chemiluminiscence, Nitric oxide electrode, Electron paramagnetic resonance (EPR) spectroscopy Indirect Determinations • using probes or spin traps Spin trap EPR, Colorimetric methods, Luminometric methods, Fluorimetric methods • determination of final product (foot prints) or consumption of substrate Oxygen electrode, HPLC, GC/MS, immunoanalysis ... Methods for determination of reactive oxygen species Direct determination of 02 consumption Electron paramagnetic resonance (EPR) spectroscopy Fluorimetric methods Colorimetric methods Luminometric methods Direct determination of 02 consumption Clark electrode Measures oxygen on a catalytic platinum surface 02 + 2 e- + 2 H20-* H202 + 2 Ohľ The electrode compartment is isolated from the reaction chamber by a thin Teflon membrane; the membrane is permeable to molecular oxygen and allows this gas to reach the cathode, where it is electrolytically reduced. The reduction allows a current to flow; this creates a potential difference which is recorded on a flatbed chart recorder. The trace is thus a measure of the oxygen activity of the reaction mixture. The current flowing is proportional to the activity of oxygen. Reference: Wikipedia - Trinity College Dublin, Biochemistry Laboratory Manual for Senior Freshman Science, 2005-2006. www.tcd.ie/biochemistry (A) Pt- (B) Ag/AgCI-electrode (C) KCl electrolyte (D) teflon membrane (E) rubber ring (F) voltage supply (G) galvanometer Electron paramagnetic resonance spectroscopy Spin traps Probe traps a radical the radical's electron spin resonance signal is destroyed and the spin trap is detected by EPR. Example: 5,5,-dimethyl -1-pyrroline-1-oxide DMPO selectively reacts with 02 and OH DMPO + 02- —► DMPO-OOH aduct DMPO + OH —► DMPO-OH aduct Fluorogenic Spin Traps TEMP0-9-AC and proxyl fluorescamine 58-61 - contain a nitroxide moiety that effectively quenches its fluorescence. However, once TEMP0-9-AC or proxyl fluorescamine traps a hydroxyl radical or superoxide, its fluorescence is restored and making these probes useful for detecting radicals either by fluorescence or by EPR. Fluorescent probes dichlorodihydrofluorescein diacetate (DCFH-DA) dihydroethidine (HE) dihydrorhodamine 123 (DHR 123) and dihydrorhodamine 6G dihydrocalcein AM Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) 3'-(p-Aminophenyl) fluorescein (APF) and 3'-(p-hydroxyphenyl) fluorescein (HPF) (pentafluorobenzoyl)aminofluorescein diacetate (PFB-H2FDA) MitoSOX Red Mitochondrial Superoxide Indicator MitoTracker Orange (CM-H2TMRos) and MitoTracker Red (CM-H2XROS) Detection: fluorometers, flow cytometers, confocal microscopes DCFH-DA 2',7'-dichlorofluorescin diacetate CH3-C- / 0-C-CH3 2',7'-dichlorofluorescin COOH • OH Cellular Esterases Hydrolysis COOH Example: Neutrophil Oxidative Burst Oxidation DCFH-DA J.P. Robinson et al. 1998 http://www.cyto.purdue.edu/ 2',7'-dichlorofluorescein Fluorescent XCI Exc. 488 rtrtu Em. 520 / COOH Control PMA-stimulated PMN Ibg green1Fluorescérfce Bovine pulmonary artery endothelial (BPAEC) cells were initially stained with the CM-H2DCFDA. After a 30-minute incubation, the cells were washed and then incubated simultaneously with FM 5-95 and Hoechst 33342 in PBS for an additional five minutes before washing and mounting. The red-fluorescent FM 5-95 appears to stain both the plasma membrane and early endosomes; the green-fluorescent, oxidized early endosomes; oxidized carboxydichlorofluorescein localizes to the cytoplasm; and the blue-fluorescent Hoechst 33342 dye stains the nucleus. Hydroethidine "NCH2CH3 ■+Br- s CH2CH3 Fluorescent Exc. 488 Em. 600 MADPJ-J UJíJdrisa Ethidium bromide 1 log red PPuoresceňíPe J.P. Robinson et al. 1998 http://www.cyto.purdue.edu/ BPAEC were incubated with weakly blue-fluorescent dihydroethidium and the green-fluorescent mitochondrial stain, MitoTracker Green FM. Upon oxidation, red-fluorescent ethidium accumulated in the nucleus. Dihydrohodamine 123 ,C—OCH3 X—OCH3 Dihydrhodamine 123 Rhodamine 123 freely permeable through cell membrane • non fluorescent • localized within mitochondria • red fluorescent Exc. 488 nm Em. 515 nm 3'-(p-hydroxyphenyl) fluorescein (HPF) and 3'-(p-aminophenyl) fluorescein (APF) X-O Hydroxyphenyl fluorescein (HPF) X=NH Amínophenyl fluorescein {A P F} ROS O X£€r Fluorescent Comparison of APF, HPF, and H2DCFDA ROS APF HPF H2DCFDA Hydrogen peroxide (H202) <1 2 190 Hydroxyl radical (HO) 1200 730 7400 Hypochlorite anion (-OCI) 3600 6 86 Nitric oxide (NO) <1 6 150 Peroxyl radical (ROO) 2 17 710 Peroxynitrite anion (ONOO-) 560 120 6600 Singlet oxygen (102) 9 5 26 Superoxide anion (O2-) 6 8 67 Autooxidation -exposure to fluorescent light <1 <1 2000 Colorimetric Methods • Cytochrome C assay The principle of the method is based on reduction of oxidized (Fe3+) cytochrome C by 02"- to form Fe2+ cytochrome C with absorption max. at 550 nm. Selective method for 02"- • Nitro b lue tetrazolium chloride (NBT) assay NBT is a potent redox indicator forming an insoluble diformazane upon reduction with absorption max. at 605 nm. NBT could be reduced by different free radicals it is not specific. Detection: spectrophotometers and histochemistry Luminometric Methods • Visible-range low-level (native) chemiluminescence • Electronically excited molecular oxygen or carbonyl groups by free radicals (chemiexcitation) to higher energy status emit weak light - chemiluminescence • Detected by ultrasensitive luminometers - high sensitive single photon counting systems • Based on wavelength of light can be selective for specific molecules (singlet oxygen 634 nm, carbonyls 500-560 nm or 375-455nm) Enhanced chemiluminiscence Enhancers/luminophores for ROS, lipid peroxide, carbonyl groups Chemiluminiscence enhanced by luminophores o VvJh + NH2 0 Luminol or •o2 . •OH H202 Oxidant OCOOH ,, , cooh+N'2+Light at NH8 *"425 nm OC -aminophthalate Most popular luminophores for ROS Luminol, Izoluminol, Lucigenin, Pholasin, Detection of CL • luminometers for cuvettes, for microplates, with chambers for whole organs • microscopes equipped with CL detection systems Typical time course of determination of ROS production (oxidative burst) of blood phagocytes by CL => 800 T cc m O 600 + Z UJ o £2 400 + 200-- S UJ S o Opsonizovaný zymosan + 15 30 Čas (min) 45 60 tu o z UJ o co UJ UJ X o 150 j 120-- 90-- Vápníkový ionophor /\ 30 Čas (min) 45 60 S 250 t _i 0£ uí 200--O Ö 150 f (O UJ S 100 + 5 1 50 + UJ 5 c PMA FMLP 15 30 Čas (min) 45 60 30 Čas (min) 45 60 Other luminophores Pholasin Luminescent protein produced by the marine rock-boring mollusc Pholas dactylus Coelenterazine Unlike luminol, coelenterazine exhibits luminescence that does not depend on the activity of cell-derived myeloperoxidase and is not inhibited by azide. MCLA Detection of superoxide. pH optimum of MCLA for luminescence generation is closer to the physiological near-neutral range than are the pH optima of luminol and lucigenin. Major sources of errors during ROS determination • presence of antioxidants (phenol red, DMSO, high concentration of proteins, ...) • presence of compounds amplifying ROS production (ions of metals, ...) presence of compounds interfering with measurement - Increasing auto-fluorescence (phenol red, ...) - Quenching luminiscence (erythrocytes, phenol red, ... ) Detection of NO Direct determination - NO electrode - fluorescent probes Indirect determination - accumulation of N02 and N03 (CL method, Griess reaction) - determination of nitrotyrosine (immunochemistry, HPLC/MS, GC/MS) ISO-NO Nitric Oxide Meter NO electrode Glass Capillary Naflon WPI Membrane / — 1 _ 1 ^H vJ - 1 vJ 0.8-7 um Carbon Fiber Insulation Size from 100 nm - 200 |jm Different sensitivity - best electrodes limit less than 0.5 nM Use for aqueous solutions, cell cultures, in vivo tissue applications Fluorescent probes 4,5-diaminofluorescein diacetate (DAF-2 diacetate) 4-amino-5-methylamino- 2',7'-difluorofluorescein (DAF-FM diacetate) DAF-FM-DA iUdaíheJjíjJ üöJJ DAť-ťM-DÁ a-narasjs DJ-.P-PM Exc. 495nm Em. 515nm 6-t-Ci^ DAF-FM di&Mteto_____* DAF-FM BttnzDtriHZDfe dftriratta Detection - fluorimeter, flowcytometer, confocal microscope Griess reaction Assay for nitrites Nitrates have to be reduced to nitrites Detection limit of about 100 nM NHCCH^Hj NHfCH^JSIHj Hazoniurn sulfanilic acid N-(1 -naphthyl)ethylenediamine purple azo derivative Abs. 548 nm Nitrite/nitrate detection by chemiluminescence N02 + 2H+ + e-NO,- + 4H+ + 3e- V(lll) V (III) NO + H20 N02 + 02+light Nitric oxide carrier flow I I 70 cc/min I c_ \ \, O*" E o > ní -o ~ o [fl programmable heater AATEK Model 745j y vacuum NO detector integrator r~ Ml lví/i"ŕĽMCOCL 74S wnnTVwrem: 4 í analyzer . I" I «^ 0 Determination of products of free radical reactions (footprints) DNA - DNA strand breaks - modified bases (e.g. 8-hydroxyguanine) - poly(ADP)polymerase activation Proteins - carbonyl groups - GSH/GSSH - changes of structure or activity Lipids - Thiobarbituric acid reactive substances - HPLC, GC - iodometries - enzymatic methods Determination of DNA strand breaks Gel electrophoresis / Pulse gel electrophoresis Comet assay lysis Schematic presentation of the comet assay Endonuclease 1 Electrophoresis Fluorimetric analysis of DNA unwinding Introducing of breaks into the back bone increase the rate of unwinding process The principle An extract of cell suspension is exposed to alkaline denaturating conditions for fixed period of time, the pH is then lowered to stop further unwinding, and the amount of residual double-stranded DNA is determined using fluorescence of ethidum bromide. Determination of modified bases Mostly quantification of 8-hydroxyadenine, 8-hydroxyguanine, thymine glycol, 8-nitroguanine, 8-oxoguanine ... Determination as the nucleoside after enzymatic hydrolysis of DNA or as the base after acid hydrolysis of DNA Analysis - HPLC, GC, thin layer chromatography - ion-mass spectrometry, nuclear magnetic resonance - determination bv ELISA carbonyl groups Reaction with dinitrophenylhydrazine (DNP) - Direct detection of product at 370 nm - immunochemistry antibodies against DNP Western blot, ELISA, immunohistochemistry Quantification of GSH and GSSH -HPLC - ione-exchange chromatography - fluorometric method e.g. o-phthaldiadehyde - enzymatic determinations Lipid peroxidation Diene conjugation - conjugated diene structures absorb ultraviolet light in the wavelength range 230-235 nm Thiobarbituric acid reactive substances The sample is boiled for 10-15 min in the presence of thiobarbituric acid under acidic conditions, and the formation of TBA-MDA adduct (pink color) measured at or close to 532 nm. TBA-MDA adduct by HPLC or gas chromatographic methods Fluorescent probes c/s-Paranaric Acid Hx_ „»H h CH3CH2 H/C_ C^C=CC^ /(CH2)7COOH BODIPY 581/591 BODIPY 665/667 Nitrotyrosine -HPLC - Anti - nitrotyrosine antibodies Determination of nitrotyrosine-containing proteins and peptide I Detection - immunohistochemistry or Western blotting ♦V BAEC treated with peroxynitrite anti-nitrotyrosine green blue-fluorescent DAPI Molecular probes, 2000 NOoTyr (|jM) 0 100 500 ■: *** ^WHW 'mgWV toHWMP NOoTyr Glu-Tub Anh Phung, 2003