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1 EPR, detection of radicals in tissue Walee Chamulitrat, Ph.D. The German Cancer Research Center (DKFZ) Applied Tumorvirology, F0700 Im Neuenheimerd Feld.

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Presentation on theme: "1 EPR, detection of radicals in tissue Walee Chamulitrat, Ph.D. The German Cancer Research Center (DKFZ) Applied Tumorvirology, F0700 Im Neuenheimerd Feld."— Presentation transcript:

1 1 EPR, detection of radicals in tissue Walee Chamulitrat, Ph.D. The German Cancer Research Center (DKFZ) Applied Tumorvirology, F0700 Im Neuenheimerd Feld 242 Heidelberg GERMANY Virtual Free Radical School EPR, detection of radicals in tissue Oxygen Society Educational Program Chamulitrat 1

2 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 2 Contents First and classical EPR detection in tissues Types of radicals detectable in tissues u Ascorbate radical u Melanin, Tyrosine radicals u Nitrosyl complexes u Organic radicals u Manganese ions EPR conditions to best study tissues u Room temperature or frozen u Direct or spin trapping Examples from EPR studies of intestinal tissues u tert-butyl (tBOOH) as a model of lipid hydroperoxides u Trinitrobenzenesulfonic acid (TNBS) as an inducer of colonic inflammation Literature Survey u Radicals in tooth and bone tissues u Radicals in skeletal muscle tissues u Radicals in other tissues

3 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 3 First and Classical EPR detection in tissues (1) n Nitrosyl complexes by Barry Commoner u Changes in electron spin resonance signals of rat liver during chemical carcinogenesis. Nature. 1965;207(3): u Electron spin resonane of iron-nitric oxide complexes with amino acids, peptides and proteins. Biochim Biophys Acta. 1968;160(3): u Isolation and identification of a paramagnetic complex from the livers of carcinogen-treated rats. Biochim Biophys Acta. 1970;201(1): u Further study on the properties of the rat liver protein involved in a paramagnetic complex in the livers of carcinogen-treated rats. Biochim Biophys Acta. 1972;257(2):

4 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 4 First and Classical EPR detection in tissues (2) n Organic radicals in tissues u Mechanically induced free radicals in bone. Nature May 4;218(140):466-7 u Electron spin resonance signals in injured nerve. Science Aug 15;165(894): u Transitory free radicals in irradiated animal tissues. Nature Sep 20;223(212): u Detection and investigation of a new type of ESR signal characteristic of some tumour tissues. Nature 1969 Apr 12;222(189):165-7 u ESR signals during x-irradiation of tissue: their characteristics and relationship to the cancerous state. Ann N Y Acad Sci. 1973;222: u Electron spin resonance of organic free radicals in dental enamel and other calcified tissues. Arch Oral Biol. 1976;21(4):

5 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 5 Types of radicals detectable in tissues (1) n Ascorbate radical u Radiosensitivity and the ascorbic acid electron spin resonance doublet. Biochim Biophys Acta ;329(1): u Study of free radicals in paraffin embedded and deparaffinized human heart muscle tissue using electron spin resonance (ESR). Histochemistry 1987;87(5): u Free radical formation in murine skin treated with tumour promoting organic peroxides. Carcinogenesis 1993;14(8): u Ascorbyl radical as natural indicator of oxidative stress: quantitative regularities. Free Radic Biol Med 1994;17(2): u Ultraviolet light-induced free radical formation in skin: an electron paramagnetic resonance study. Photochem Photobiol 1994 Jan;59(1):1-4 u Exercise training generates ascorbate free radical in rat heart. Indian J Physiol Pharmacol 1995;39(4):323-9 u Detection of an increase in ascorbate radical in an irradiated experimental tumour system using ESR. Int J Radiat Biol 1995 ;68(4): u Detection of an ascorbate radical in an irradiated mice using electron spin resonance (ESR). Indian J Exp Biol 1996 Sep;34(9): Increase in production of ascorbate radical in tissues of rat treated with paraquat. Free Radic Res 2000;33(2):179-85

6 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 6 Types of radicals detectable in tissues (2) n Melanin and tyrosine radicals u EPR investigations of the iron domain in neuromelanin. Biochim Biophys Acta 1997;1361(1):49-58 u Binding of iron to neuromelanin of human substantia nigra and synthetic melanin: an electron paramagnetic resonance spectroscopy study. Free Radic Biol Med 1997;23(1):110-9 u Q-band EPR investigations of neuromelanin in control and Parkinson's disease patients. Biochim Biophys Acta 2000;1500(3): u Ribonucleotide reductase in melanoma tissue. EPR detection in human amelanotic melanoma and quenching of the tyrosine radical by 4-hydroxyanisole. J Cancer Res Clin Oncol 1991;117(2):91-5 n Nitrosyl iron complexes u Many investigators have utilized EPR for nitric oxide detection in blood and tissues since mid 1980s after the discovery of physiological role of nitric oxide. To name a few, these investigators are Y. Henry, A.F. Vanin, A. Muelsch, J.R. Lancaster, J. Hibbs, T. Akaike, H. Maeda, W. Chamulitrat, T. Ohnishi, T. Yonetani, and J. Zweier.

7 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 7 Types of radicals detectable in tissues (3) Organic radicals u Symons MC. Radicals generated by bone cutting and fracture. Free Radic Biol Med 1996;20(6):831-5 An electron spin resonance study on alkylperoxyl radical in thin-sliced renal tissues from ferric nitrilotriacetate-treated rats: the effect of alpha- tocopherol feeding. Free Radic Res. 2001;35(3): n Manganese ions u Paramagnetic changes in cancer: DMBA-induced tumours studied in non- lyophilized and lyophilized tissues. Br J Cancer 1979;39(3):330-6 u Partition of divalent and total manganese in organs and subcellular organelles of MnCl 2 -treated rats studied by ESR and neutron activation analysis. Biochim Biophys Acta 1985;841(2):208-14

8 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 8 EPR conditions to best study tissues (1) Room temperature RT measurement can be done using a quartz tube (for tooth/bone samples) and a tissue EPR flat cell (for wet tissue). Organic radicals are normally detected at RT. Mn +2 may also be detected with six broad lines. For wet tissue, radicals from exogenously added precursors (e.g., chemical, redox cyclers), and spin trap adducts (for spin trapping experiment), can be easily detectable. Frozen measurement Liquid nitrogen temperature measurement is commonly used to detect metal centers with an unpaired electron such as nitrosyl iron complexes (g~2.04, and g ani ~ ), iron (III) (e.g., cytochrome p450s in liver, g~6), Fe(III)-desferal (g~4.3), Mn +2 (6 lines), and Cu +2 (g ani at ). It depends on what radicals one wants to measure !

9 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 9 EPR conditions to best study tissues (2) Direct EPR n High fluxes of radical generation have to be produced. This can be achieved from tissue metabolism of a compound by adding it directly to tissue, and measure. n At optimal conditions, ascorbate radical can be directly detected, e.g., with UV exposure of mouse skin, or treatment with peroxides or redox cyclers. Spin trapping n Spin trapping is appropriate to detect diatomic, triatomic radicals such as, hydoxyl, superoxide, sulfite radicals as well as lipid/carbon centered radicals from the tissue itself. Spin trap can be direclty added to tissue or topically applied on mouse skin (prior to a stress treatment). DMPO, DEPMPO, 4- POBN have been used in tissue and skin of lab animals. It depends on what radicals one wants to measure !

10 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 10 Antioxid Redox Signal 3, , ascorbate radical g ~ g ~ A B C 20 Gauss Ileum tBOOH Ileum A - B Peroxyl radical formation in the rat ileum Metabolism of tert -BOOH by Intestinal tissues Rat ileum, with an aliquot ot tert -BOOH, was placed into a flat cell, and examined by EPR at room temperature. Examples from EPR studies of intestinal tissues

11 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 11 Intestinal cells produce nitric oxide under pathophysiological conditions 80 Gauss a = 17 G ZZ jejunum of endotoxic rat Life Sci. 57, , Shock 5, 59-65, Nitrosyl heme and non-heme formation in intestinal tissues measured at 77K.

12 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 12 Scraped intestinal tissues from LPS-treated rats produced decreased amounts of tBOOH-derived radicals untreated ratLPS-treated rat 80 Gauss 20 Gauss Nitrosyl hemoglobin tBOOH-derived radicals Blood Intestinal tissue Antioxid Redox Signal 3, , Spin trapping of tBOOH-derived radicals by DMPO (Room temperature measurement).

13 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 13 duodenum TNBS duodenum BBA 1336, 73-82, 1997 Metabolism of TNBS by intestinal tissues measured at room temperature 10 Gauss

14 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 14 A + SOD A with heat-killed cells 10 Gauss enterocytes NADPH TNBS DMPO * * * * * * A NADPH NADP + F FH 2 FH RNO 2 O 2 BBA 1336, 73-82, Redox cycling of TNBS produces superoxide radical by enterocytes, colonocytes, and red blood cells when diluted cell suspension (2 x 10 6 cells/ml) was used. RNO 2 O 2

15 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 15 BBA 1472, , H SO 3 2 [O] SO 3 SO H 3 NO 2 O N 2 H H NO H SO 32 NO 2 O N 2 H H NO 2 NH R 2 NH R Desulfonation of TNBS proceeds with high protein concentrations Oxidation of sulfite produces sulfite radical Sulfite radical is trapped by DMPO, or SO 3 = adds directly to DMPO.

16 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat Gauss 50%RBC's TNBS DMPO 50%RBC's TNBS DEPMPO TNBS TNBS-derived sulfite radical generation by red blood cells At high red blood cell concentrations, sulfite radical was produced as trapped spin trap DMPO or DEPMPO. DEPMPO sulfite adduct was more persistent.

17 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat Gauss A B C colonic mucosa TNBS DMPO colonic mucosa TNBS A - B ascorbate radical TNBS-derived sulfite radical generation by colonic mucosa Thick mucosal tissues produced sulfite radical from TNBS.

18 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat Gauss colonic mucosa TNBS submucosal muscle layer TNBS * * * * * * * * * = TNBS * * * * * * Intestinal submucosal muscle layer produces significant levels of sulfite radical from TNBS One can measure the extent of TNBS metabolism from different tissue preparations.

19 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 19 Radicals detected in tooth and bone tissues (1) n Monte Carlo calculations of the dose distribution in teeth due to internal exposure from 90 Sr: application to EPR tooth dosimetry. Radiat Prot Dosimetry. 2001; 93(3): n ESR dosimetry of 89 Sr and 153 Sm in bone. Appl Radiat Isot Feb;54(2): n From dating to biophysics--20 years of progress in applied ESR spectroscopy. Appl Radiat Isot May; 52(5): n Relationships between ESR-evaluated doses estimated from enamel and activity of radionuclides in bone and teeth of reindeer. Appl Radiat Isot Mar; 50(3): n Heme compounds in dinosaur trabecular bone. Proc Natl Acad Sci USA Jun 10; 94(12):

20 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 20 Radicals detected in tooth and bone tissues (2) n Proton dosimetry in bone using electron spin resonance. Appl Radiat Isot Nov-Dec; 47(11-12): n Radiation dosimetry of an accidental overexposure using EPR spectrometry and imaging of human bone. Appl Radiat Isot Nov-Dec; 47(11-12): n Radicals generated by bone cutting and fracture. Free Radic Biol Med. 1996; 20(6): Review. n Radiation dosimetry in human bone using electron paramagnetic resonance. Phys Med Biol Dec; 40(12):

21 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 21 Radicals detected in skeletal muscle tissues n Enhanced generation of reactive oxygen species in the limb skeletal muscles from a murine infarct model of heart failure. Circulation Jul 10; 104(2): n Detection of reactive oxygen and reactive nitrogen species in skeletal muscle. Microsc Res Tech Nov 15; 55(4): Review. n Electron paramagnetic resonance: a high-resolution tool for muscle physiology. Exerc Sport Sci Rev. 2001; 29(1):3-6. Review. n Electron paramagnetic resonance reveals age-related myosin structural changes in rat skeletal muscle fibers. Am J Physiol Cell Physiol Mar; 280(3):C n Electron paramagnetic resonance identification of irradiated cuttlefish (Sepia officinalis L.) Appl Radiat Isot May; 52(5):

22 EPR, detection of radicals in tissueOxygen Society Educational ProgramChamulitrat 22 Radicals in other tissues n Electron resonance studies on the influence of anionic surfactants on human skin. Dermatology. 1997; 194(3): n Direct current shocks to the heart generate free radicals: an electron paramagnetic resonance study. J Am Coll Cardiol Nov 15; 28(6): n A technique for the fast sampling of biological tissues for electron paramagnetic resonance spectroscopy. (in rabbit spinal cord) Free Radic Biol Med. 1996; 20(1): n Detection of an increase in ascorbate radical in an irradiated experimental tumour system using ESR. Int J Radiat Biol Oct; 68(4):


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