1 / 22

EPR, detection of radicals in tissue

Virtual Free Radical School. EPR, detection of radicals in tissue. Walee Chamulitrat, Ph.D. The German Cancer Research Center (DKFZ) Applied Tumor Virology, F0700 Im Neuenheimerd Feld 242 Heidelberg GERMANY 049-06221-424834 w.chamulitrat@dkfz-heidelberg.de.

gtolbert
Download Presentation

EPR, detection of radicals in tissue

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Virtual Free Radical School EPR, detection of radicals in tissue Walee Chamulitrat, Ph.D. The German Cancer Research Center (DKFZ) Applied Tumor Virology, F0700 Im Neuenheimerd Feld 242 Heidelberg GERMANY 049-06221-424834 w.chamulitrat@dkfz-heidelberg.de EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine Chamulitrat 1

  2. Contents First and classical EPR detection in tissues Types of radicals detectable in tissues • Ascorbate radical • Melanin, Tyrosine radicals • Nitrosyl complexes • Organic radicals • Manganese ions EPR conditions to best study tissues • Room temperature or frozen • Direct or spin trapping Examples from EPR studies of intestinal tissues • tert-butyl (tBOOH) as a model of lipid hydroperoxides • Trinitrobenzenesulfonic acid (TNBS) as an inducer of colonic inflammation Literature Survey 1995-2002 • Radicals in tooth and bone tissues • Radicals in skeletal muscle tissues • Radicals in other tissues Society For Free Radical Biology and Medicine

  3. First and Classical EPR detection in tissues (1) • Nitrosyl complexes by Barry Commoner • Changes in electron spin resonance signals of rat liver during chemical carcinogenesis. Nature. 1965; 207(3):1246-9. • Electron spin resonance of iron-nitric oxide complexes with amino acids, peptides and proteins. Biochim Biophys Acta.1968; 160(3):311-20. • Isolation and identification of a paramagnetic complex from the livers of carcinogen-treated rats. Biochim Biophys Acta. 1970; 201(1):131-40. • 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):452-60. Society For Free Radical Biology and Medicine

  4. First and Classical EPR detection in tissues (2) • Organic radicals in tissues • Mechanically induced free radicals in bone. Nature. 1968 May 4; 218(140):466-7 • Electron spin resonance signals in injured nerve. Science. 1969 Aug 15; 165(894):703-4. • Transitory free radicals in irradiated animal tissues. Nature. 1969 Sep 20; 223(212):1229-33. • Detection and investigation of a new type of ESR signal characteristic of some tumour tissues. Nature1969 Apr 12; 222(189):165-7 • ESR signals during x-irradiation of tissue: their characteristics and relationship to the cancerous state. Ann N Y Acad Sci. 1973; 222:1077-86. • Electron spin resonance of organic free radicals in dental enamel and other calcified tissues. Arch Oral Biol.1976; 21(4): 227-32. Society For Free Radical Biology and Medicine

  5. Types of radicals detectable in tissues (1) • Ascorbate radical • Radiosensitivity and the ascorbic acid electron spin resonance doublet. Biochim Biophys Acta. 1973; 329(1):159-62. • Study of free radicals in paraffin embedded and deparaffinized human heart muscle tissue using electron spin resonance (ESR). Histochemistry 1987; 87(5):499-504 • Free radical formation in murine skin treated with tumour promoting organic peroxides. Carcinogenesis 1993; 14(8):1499-503 • Ascorbyl radical as natural indicator of oxidative stress: quantitative regularities. Free Radic Biol Med 1994; 17(2):93-103 • Ultraviolet light-induced free radical formation in skin: an electron paramagnetic resonance study. Photochem Photobiol 1994 Jan; 59(1):1-4 • Exercise training generates ascorbate free radical in rat heart. Indian J Physiol Pharmacol 1995; 39(4):323-9 • Detection of an increase in ascorbate radical in an irradiated experimental tumour system using ESR. Int J Radiat Biol 1995; 68(4):467-73 • Detection of an ascorbate radical in an irradiated mice using electron spin resonance (ESR). Indian J Exp Biol 1996; 34(9):898-900 • Increase in production of ascorbate radical in tissues of rat treated with paraquat. Free Radic Res 2000; 33(2):179-85 Society For Free Radical Biology and Medicine

  6. Types of radicals detectable in tissues (2) • Melanin and tyrosine radicals • EPR investigations of the iron domain in neuromelanin. Biochim Biophys Acta 1997;1361(1):49-58 • 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 • Q-band EPR investigations of neuromelanin in control and Parkinson's disease patients. Biochim Biophys Acta 2000;1500(3):306-12 • Ribonucleotide reductase in melanoma tissue. EPR detection in human amelanotic melanoma and quenching of the tyrosine radical by 4-hydroxyanisole. JCancer Res Clin Oncol 1991;117(2):91-5 • Nitrosyl iron complexes • Many investigators have utilized EPR for nitric oxide detection in blood and tissues since mid 1980’s 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, G.R. Buettner and J. Zweier. Society For Free Radical Biology and Medicine

  7. Types of radicals detectable in tissues (3) • Organic radicals • 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 Rad Res. 2001; 35(3):245-55 • Manganese ions • Paramagnetic changes in cancer: DMBA-induced tumours studied in non-lyophilized and lyophilized tissues. Br J Cancer 1979; 39(3):330-6 • Partition of divalent and total manganese in organs and subcellular organelles of MnCl2-treated rats studied by ESR and neutron activation analysis. Biochim Biophys Acta 1985; 841(2):208-14 Society For Free Radical Biology and Medicine

  8. EPR conditions to best study tissues (1) It depends on what radicals one wants to measure ! 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 gani ~2.1-2.0), iron (III) (e.g., cytochrome p450‘s in liver, g~6), Fe(III)-desferal (g~4.3), Mn2+ (6 lines), and Cu+2 (gani at 2.1-2.0). 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. Mn2+ may also be detected with six broad lines (span over 600 G). 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. Society For Free Radical Biology and Medicine

  9. EPR conditions to best study tissues (2) It depends on what radicals one wants to measure ! Direct EPR • 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. • 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 • 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. Society For Free Radical Biology and Medicine

  10. P e r o x y l r a d i c a l f o r m a t i o n i n t h e r a t i l e u m a s c o r b a t e r a d i c a l g ~ 2 . 0 1 3 I l e u m t B O O H I l e u m g ~ 2 . 0 1 3 A - B 2 0 G a u s s Examples from EPR studies of intestinal tissues Metabolism of tert-BOOH by Intestinal tissues A Rat ileum, with an aliquot ot tert-BOOH, was placed into a flat cell, and examined by EPR at room temperature. B C Antiox Redox Signal 3, 177-187, 2001. Society For Free Radical Biology and Medicine

  11. jejunum of endotoxic rat 8 0 G a u s s a = 1 7 G Z Z Intestinal cells produce nitric oxide under pathophysiological conditions Nitrosyl heme and non-heme formation in intestinal tissues measured at 77 K. Life Sci. 57, 387-395, 1995. Shock 5, 59-65, 1996. Society For Free Radical Biology and Medicine

  12. Scraped intestinal tissues from LPS-treated rats produced decreased amounts of tBOOH-derived radicals 80 Gauss 20 Gauss untreated rat LPS-treated rat Blood Nitrosyl hemoglobin Intestinal tissue Spin trapping of tBOOH-derived radicals by DMPO (Room temperature measurement). tBOOH-derived radicals Antioxid Redox Signal 3, 177-187, 2001. Society For Free Radical Biology and Medicine

  13. d u o d e n u d u o d e n u m T N B S Metabolism of TNBS by intestinal tissues measured at room temperature m BBA 1336, 73-82, 1997 10Gauss Society For Free Radical Biology and Medicine

  14. + F H N A D P R N O 2 2 F H O 2 e n t e r o c y t e s N A D P H T N B S D M P O A + S O D A w i t h h e a t - k i l l e d c e l l s 1 0 G a u s s O2 Redox cycling of TNBS produces superoxide radical by enterocytes, colonocytes, and red blood cells when diluted cell suspension (2 x 106 cells/mL) was used. RNO2 F N A D P H * * * A * * * BBA 1336, 73-82, 1997. Society For Free Radical Biology and Medicine

  15. R N H R S O H 3 O N N O O N N O 2 2 2 + 2 N H H S O + 2 3 2 H H H H N O N O 2 2 [ O ] H S O S O 2 3 3 Desulfonation of TNBS proceeds with high protein concentrations Oxidation of sulfite produces sulfite radical Sulfite radical is trapped by DMPO, or SO32- adds directly to DMPO. BBA 1472 , 368-375, 1999. Society For Free Radical Biology and Medicine

  16. TNBS-derived sulfite radical generation by red blood cells 5 0 % R B C ' s T N B S D M P O 5 0 % R B C ' s T N B S D E P M P O T N B S 2 0 G a u s s At high red blood cell concentrations, sulfite radical was produced as trapped spin trap DMPO or DEPMPO. DEPMPO sulfite adduct was more persistent. Society For Free Radical Biology and Medicine

  17. TNBS-derived sulfite radical generation by colonic mucosa a s c o r b a t e r a d i c a l c o l o n i c m u c o s a T N B S D M P O A B c o l o n i c m u c o s a T N B S C A - B 1 0 G a u s s Thick mucosal tissues produced sulfite radical from TNBS. Society For Free Radical Biology and Medicine

  18. = T N B S c o l o n i c m u c o s a T N B S * * * * * * * * * * s u b m u c o s a l m u s c l e l a y e r T N B S * * * * * 1 0 G a u s s Intestinal submucosal muscle layer produces significant levels of sulfite radical from TNBS One can measure the extent of TNBS metabolism from different tissue preparations. Society For Free Radical Biology and Medicine

  19. Radicals detected in tooth and bone tissues (1) • Monte Carlo calculations of the dose distribution in teeth due to internal exposure from 90Sr: application to EPR tooth dosimetry. Radiat Prot Dosimetry. 2001; 93(3):245-60. • ESR dosimetry of 89Sr and 153Sm in bone. Appl Radiat Isot. 2001 Feb;54(2):269-74. • From dating to biophysics--20 years of progress in applied ESR spectroscopy. Appl Radiat Isot. 2000 May; 52(5):1023-30. • Relationships between ESR-evaluated doses estimated from enamel and activity of radionuclides in bone and teeth of reindeer. Appl Radiat Isot. 1999 Mar; 50(3):567-72. • Heme compounds in dinosaur trabecular bone. Proc Natl Acad Sci USA. 1997 Jun 10; 94(12):6291-6. Society For Free Radical Biology and Medicine

  20. Radicals detected in tooth and bone tissues (2) • Proton dosimetry in bone using electron spin resonance. Appl Radiat Isot. 1996 Nov-Dec; 47(11-12):1533-8. • Radiation dosimetry of an accidental overexposure using EPR spectrometry and imaging of human bone. Appl Radiat Isot. 1996 Nov-Dec; 47(11-12):1345-50. • Radicals generated by bone cutting and fracture. Free Radic Biol Med. 1996; 20(6):831-5. Review. • Radiation dosimetry in human bone using electron paramagnetic resonance. Phys Med Biol. 1995 Dec; 40(12):2065-77. Society For Free Radical Biology and Medicine

  21. Radicals detected in skeletal muscle tissues • Enhanced generation of reactive oxygen species in the limb skeletal muscles from a murine infarct model of heart failure. Circulation. 2001 Jul 10; 104(2):134-6. • Detection of reactive oxygen and reactive nitrogen species in skeletal muscle. Microsc Res Tech. 2001 Nov 15; 55(4):236-48. Review. • Electron paramagnetic resonance: a high-resolution tool for muscle physiology. Exerc Sport Sci Rev. 2001; 29(1):3-6. Review. • Electron paramagnetic resonance reveals age-related myosin structural changes in rat skeletal muscle fibers. Am J Physiol Cell Physiol. 2001 Mar; 280(3):C540-7. • Electron paramagnetic resonance identification of irradiated cuttlefish (Sepia officinalis L.) Appl Radiat Isot. 2000 May; 52(5):1385-90. Society For Free Radical Biology and Medicine

  22. Radicals in other tissues • Electron resonance studies on the influence of anionic surfactants on human skin. Dermatology. 1997; 194(3):238-42. • Direct current shocks to the heart generate free radicals: an electron paramagnetic resonance study. J Am Coll Cardiol. 1996 Nov 15; 28(6):1598-609. • 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):89-91. • Detection of an increase in ascorbate radical in an irradiated experimental tumour system using ESR. Int J Radiat Biol. 1995 Oct; 68(4):467-73. Society For Free Radical Biology and Medicine

More Related