Virtual Free Radical School EPR detection of radicals

Virtual Free Radical School EPR, detection of radicals in tissue Walee Chamulitrat, Ph. D. The German Cancer Research Center (DKFZ) Applied Tumor Virology, F 0700 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 1 Chamulitrat 1

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

First and Classical EPR detection in tissues (1) n Nitrosyl complexes by Barry Commoner Changes in electron spin resonance signals of rat liver during chemical carcinogenesis. Nature. 1965; 207(3): 1246 -9. u Electron spin resonance of iron-nitric oxide complexes with amino acids, peptides and proteins. Biochim Biophys Acta. 1968; 160(3): 311 -20. u Isolation and identification of a paramagnetic complex from the livers of carcinogen-treated rats. Biochim Biophys Acta. 1970; 201(1): 131 -40. 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; Society For Free Radical Biology and Medicine 257(2): 452 -60. EPR, detection of radicals in tissue u 3

First and Classical EPR detection in tissues (2) n Organic radicals in tissues Mechanically induced free radicals in bone. Nature. 1968 May 4; 218(140): 466 -7 u Electron spin resonance signals in injured nerve. Science. 1969 Aug 15; 165(894): 703 -4. u Transitory free radicals in irradiated animal tissues. Nature. 1969 Sep 20; 223(212): 1229 -33. 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: 1077 -86. u Electron spin resonance of organic free radicals in dental enamel and other Society calcified tissues. Arch Biol. 1976; For Free Radical Biology and. Oral Medicine EPR, detection of radicals in tissue 4 21(4): 227 -32. u

Types of radicals detectable in tissues (1) n Ascorbate radical u u u u u 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 EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 5

Types of radicals detectable in tissues (2) n Melanin and tyrosine radicals u u n 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. J Cancer Res Clin Oncol 1991; 117(2): 91 -5 Nitrosyl iron complexes u 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 EPR, detection of radicals in tissue 6

Types of radicals detectable in tissues (3) n Organic radicals u u n 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 u u 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 Mn. Cl 2 -treated rats studied by ESR and neutron activation analysis. Biochim Biophys Acta 1985; 841(2): 208 -14 Society For Free Radical Biology and Medicine EPR, detection of radicals in tissue 7

EPR conditions to best study tissues (1) It depends on what radicals one wants to measure ! Room temperature Frozen measurement RT measurement can be done Liquid nitrogen temperature using a quartz tube (for measurement is commonly tooth/bone samples) and a used to detect metal centers tissue EPR flat cell (for wet with an unpaired electron tissue). Organic radicals are such as normally detected at RT. nitrosyl iron complexes 2+ Mn may also be detected (g~2. 04, and gani ~2. 1 -2. 0), with six broad lines (span iron (III) (e. g. , cytochrome over 600 G). p 450‘s in liver, g~6), Fe(III)For wet tissue, radicals from desferal (g~4. 3), Mn 2+ (6 exogenously added lines), and Cu+2 (gani at 2. 1 precursors (e. g. , chemical, 2. 0). redox cyclers), and spin trap adducts (for spin trapping experiment), can be easily Society For Free Radical Biology and Medicine EPR, detection of radicals in tissue 8 detectable.

EPR conditions to best study tissues (2) It depends on what radicals one wants to measure ! 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. EPR, detection of radicals in tissue 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. n 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 9

Examples from EPR studies of intestinal Peroxyl radical formation in the rat ileum g ~ 2. 013 ascorbate radical Metabolism of tert-BOOH by Intestinal Atissues Rat ileum, with an aliquot ot tert-BOOH, was aced into a flat cell, and examined by EPR at room temperature. Ileum t. BOOH Ileum B g ~ 2. 013 A-B C Antiox Redox Signal 3, 177 -187, 2001. EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 20 Gauss 10

Intestinal cells produce nitric oxide under pathophysiological conditions itrosyl heme and non-heme rmation in intestinal tissues measured at 77 K. jejunum of endotoxic rat 80 Gauss a ZZ = 17 G Life Sci. 57, 387 -395, 1995. Shock 5, 59 -65, 1996. EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 11

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

abolism of TNBS by intestinal tissues measured at room temperature duodenum TNBS BBA 1336, 73 -82, 1997 10 Gauss EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 13

NADP+ ox cycling of TNBS produces roxide radical by enterocytes, onocytes, and red blood cells when diluted cell suspension (2 x 106 cells/m. L) was used. FH 2 RNO 2 O 2 FH F NADPH enterocytes NADPH TNBS DMPO A * * * A + SOD 10 Gauss BBA 1336, 73 -82, 1997. EPR, detection of radicals in tissue A with heat-killed cells Society For Free Radical Biology and Medicine 14

Desulfonation of TNBS proceeds with high protein concent O 2 N SO 3 H NO 2 H H + R NH 2 O 2 N NO 2 H H + H 2 SO 3 NO 2 Oxidation of sulfite produces sulfite radical [O] H 2 SO 3 Sulfite radical is trapped by DMPO, or SO 32 - adds directly t BBA 1472 , 368 -375, 1999. EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 15

TNBS-derived sulfite radical generation by red blood cells high red blood cell concentrations, te radical was produced as trapped spin trap DMPO or DEPMPO. 50%RBC's TNBS DMPO DEPMPO sulfite adduct was more persistent. 50%RBC's TNBS DEPMPO TNBS 20 Gauss EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 16

TNBS-derived sulfite radical generation by colonic mucosa Thick mucosal tissues roduced sulfite radical from TNBS. ascorbate radical A B C colonic mucosa TNBS DMPO colonic mucosa TNBS A-B 10 Gauss EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 17

Intestinal submucosal muscle layer produces significant levels of sulfite radical from TNBS * * = TNBS * * * colonic mucosa TNBS * * * One can measure the extent of TNBS metabolism from fferent tissue preparations. submucosal muscle layer TNBS * ** * * 10 Gauss EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 18

Radicals detected in tooth and bone tissues (1) n n 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): 245 -60. ESR dosimetry of 89 Sr and 153 Sm 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. EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 19

Radicals detected in tooth and bone tissues (2) n n 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. EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 20

Radicals detected in skeletal muscle tissues n n n 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): C 540 -7. Electron paramagnetic resonance identification of irradiated cuttlefish (Sepia officinalis L. ) Appl Radiat Isot. 2000 May; 52(5): 1385 -90. EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 21

Radicals in other tissues n n 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. EPR, detection of radicals in tissue Society For Free Radical Biology and Medicine 22