Transcript Anchorage Independence Growth

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
[email protected]
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
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Ascorbate radical
Melanin, Tyrosine radicals
Nitrosyl complexes
Organic radicals
Manganese ions
EPR conditions to best study tissues
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Room temperature or frozen
Direct or spin trapping
Examples from EPR studies of intestinal tissues
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tert-butyl (tBOOH) as a model of lipid hydroperoxides
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Trinitrobenzenesulfonic acid (TNBS) as an inducer of colonic inflammation
Literature Survey 1995-2002
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Radicals in tooth and bone tissues
Radicals in skeletal muscle tissues
Radicals in other tissues
EPR, detection of radicals in tissue
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First and Classical EPR detection in tissues (1)
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Nitrosyl complexes by Barry Commoner
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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.
EPR, detection of radicals in tissue
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First and Classical EPR detection in tissues (2)
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Organic radicals in tissues
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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. Nature 1969 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; 222:107786.
Electron spin resonance of organic free radicals in dental enamel and
other calcified tissues. Arch Oral Biol. 1976; 21(4): 227-32.
EPR, detection of radicals in tissue
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Types of radicals detectable in tissues (1)
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Ascorbate radical
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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
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Types of radicals detectable in tissues (2)
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Melanin and tyrosine radicals
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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
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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.
EPR, detection of radicals in tissue
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Types of radicals detectable in tissues (3)
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Organic radicals
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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 alphatocopherol feeding. Free Rad Res. 2001; 35(3):245-55
Manganese ions
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Paramagnetic changes in cancer: DMBA-induced tumours studied in nonlyophilized 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
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EPR conditions to best study tissues (1)
It depends on what radicals one wants to measure !
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.
EPR, detection of radicals in tissue
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).
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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.
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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
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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.
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Spin trap can be direclty added to
tissue or topically applied on
mouse skin (prior to a stress
treatment). DMPO, DEPMPO, 4POBN have been used in tissue
and skin of lab animals.
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Examples from EPR studies of intestinal tissues
Peroxyl rad ical form atio n in the rat ileum
ascorbate radical
g ~ 2.013
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.
Ileum
tBOOH
A
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
Chamulitrat 10
Intestinal cells produce nitric oxide under
pathophysiological conditions
Nitrosyl heme and non-heme
formation 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
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Scraped intestinal tissues from LPS-treated rats produced
decreased amounts of tBOOH-derived radicals
untreated rat
LPS-treated rat
Blood
Nitrosyl hemoglobin
Intestinal tissue
80 Gauss
Spin trapping of tBOOH-derived radicals
by DMPO (Room temperature measurement).
tBOOH-derived radicals
Antioxid Redox Signal 3, 177-187, 2001.
20 Gauss
EPR, detection of radicals in tissue
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Metabolism of TNBS by intestinal tissues
measured at room temperature
duodenum
SO3H
O2N
NO2
H
H
NO2
a N4 = 9.7 G (1)
a H3,5 = 3.2 G (2)
duodenum
TNBS
a N2,6 = 0.25 G (2)
BBA 1336, 73-82, 1997
10 Gauss
EPR, detection of radicals in tissue
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FH2
NADP+
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
O2
RNO2
O2
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
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Desulfonation of TNBS proceeds with high protein concentrations
SO3H
O2N
NO2
H
H
+
R
NH
R
NH2
O2N
NO2
H
H
+ H2SO3
NO2
NO2
Oxidation of sulfite produces sulfite radical
[O]
H2SO3
SO3
Sulfite radical is trapped by DMPO, or SO32- adds directly to DMPO.
BBA 1472 , 368-375, 1999.
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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.
50%RBC's
TNBS
DMPO
DEPMPO sulfite adduct was more
persistent.
50%RBC's
TNBS
DEPMPO
TNBS
20 Gauss
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TNBS-derived sulfite radical generation by colonic
mucosa
Thick mucosal tissues
produced sulfite radical
from TNBS.
ascorbate radical
A
B
C
colonic mucosa
TNBS
DMPO
colonic mucosa
TNBS
A-B
10 Gauss
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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
different tissue preparations.
submucosal
muscle layer
TNBS
*
**
* *
10 Gauss
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Radicals detected in tooth and
bone tissues (1)
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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.
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Radicals detected in tooth and
bone tissues (2)
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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.
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Radicals detected in skeletal
muscle tissues
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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.
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Radicals in other tissues
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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.
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