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Transcript of Dario C. Ramirez [email protected] Free Radical Metabolites Workgroup LPC/NIEHS/NIH/DHHS ...
Dario C. Ramirez
Free Radical Metabolites Workgroup
LPC/NIEHS/NIH/DHHS
http://www.niehs.nih.gov
Ramirez DC, 1 of 21
Immuno-spin Trapping of Protein and DNA Radicals
Oxidative Damage to Macromolecules
Macromolecule
Free radicals
Oxidized products
Pro-oxidants
Antioxidants
Repaired
Accumulated
Degradated
Ramirez DC, 2 of 21
Trapping of Radicals with DMPO Trapping of Radicals with DMPO
Mason, R.P. 2004. Free Radic. Biol. Med. 36: 1214-1223.
N
O
N
N
N
OH
OO
R
H
H
H
RR
RR
RR
H+
+ +
+reduction potential –1.92V
oxidation potential +1.63V
oxidation
reduction
dispropor-
tionation
(DMPO)radical adduct
nitrone adductnitrone adduct
hydroxylamine adduct
.
.
5,5-dimethyl-1-pyrroline N-oxide
Ramirez DC, 3 of 21
The specificity of the reaction of nitrone spin traps with free radicals has already made spin trapping with ESR detection the most universal,
specific tool for the detection of free radicals in biological systems.
Heterogeneous Immuno-Spin trapping Assays
. Enzyme Linked Immuno-Sorbent Assay (ELISA)
. Western blot or Immunoblot/ting
. Immuno-Dot/Slot Blot
Solid support
DMPO ( )-protein/DNA nitrone adducts
Anti-DMPO antibody
Secondary antibody conjugated to an enzyme
Substrate
Product
Ramirez DC, 4 of 21
In vitro and in vivo Production and Detection of
DNA Nitrone Adducts
Ramirez DC, 5 of 21
Ramirez, DC & Mason R.P. Improved Immuno-spin Trapping Analyses of DNA Radicals. Nature Protocols. Coming soon. Fall 2006. This article will bring to Free radical researcher’s bench a step-by-step protocol to detect DNA radicals in tissues, whole cells, organelles, and in chemical systems.
• Hydroxyl radical react with DNA (~109 M-1 s-1)
• abstraction
• addition
• DNA radicals are involved
• C- and N-centered radicals in bases (purines and pyrimidines)
• C-centered radicals in 2'-deoxyribose
• Final consequences
• base modifications
• abasic sites
• strand breaks
• tandem-base lesions
• DNA-protein cross-links
Reaction of Hydroxyl Radical with DNAReaction of Hydroxyl Radical with DNA
Evans, M.D., Dizdaroglu, M. & Cooke, M. S. 2004. Mutation Research, 567: 1-61.
Von Sonntag, C. 1987, The Chemical Basis of Radiation Biology. Tylor & Francis, London, NY.
Ramirez DC, 6 of 21
Why Develop New Technology to Study Why Develop New Technology to Study Oxidative DNA Damage? Oxidative DNA Damage?
Halliwell, B. & Whiteman, M. 2005. Br. J. Pharmacol. 142: 231-255
Collins, A.R. et al. 2004. Arch. Biochem. Biophys. 423: 57-65
Ramirez DC, 7 of 21
1. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG)
. A biomarker of oxidative DNA damage
. Misreading of 8-oxo-dG can lead to G:C T:A transversion
2. Artifactual 8-oxo-dG formation during
. isolation of DNA
. sample work-up
. analysis
3. The European Standards Committee on Oxidative DNA Damage (ESCODD) recommended the development of new approaches to detect oxidative DNA damage to bypass the artifactual oxidation of DNA in intact cells.
DNA Oxidation and its InvestigationDNA Oxidation and its Investigation
- e–, - H+
DNA radical adducts
DNA nitrone adducts
Immuno-spin TrappingImmuno-spin Trapping
DMPO
Ramirez DC, 8 of 21
Cadet, J. 1999. Hydroxyl radicals and DNA base damage. Mutat. Res. 424: 9-21
Electron
transfer
processes
DNA radicalsDNA radicals
ElectrophoresisElectrophoresis
Cleavage of phosphodiester bonds
DNA-(sugar)-centeredradicals
Immunochemical and Immunochemical and ChromatographicChromatographic
(HPLC-MS/MS, GC/MS, etc.)(HPLC-MS/MS, GC/MS, etc.) measurementmeasurement
DNA-[8-oxo-dG]
(8-oxo-7,8-dihydro-2’-
deoxyguanosine)
DNA-[8-oxo-dG]•
(8-hydroxy-7,8-dihydro-2’-
deoxyguanosyl radical)
O2
- e–, - H+
Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127
DNA Radicals, 8-oxo-dG, and Site-specific DNA Radicals, 8-oxo-dG, and Site-specific FragmentationFragmentation
500
1,0
00
Imm
un
ore
act
ivit
y (R
LU
)
0
1000
2000
3000
40008-oxo-dGnitrone adducts
0
0.5 1 5
10
50
100
[H2O2] (M)
* ** *
*
*
*
*
* **
Ramirez DC, 9 of 21
DN
A +
H2O
2
+ 1
,000
[H2O2] (M)
+ 5
00
+ 1
00
+ 5
0+
10
+ 5
DN
A +
Cu
(II)
DN
A
Agarose gel electrophoresis
Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127
[DMPO] (mM)
Imm
un
ore
act
ivit
y (R
LU
)
0 100 200 300 400 5000
1000
2000
3000nitrone adducts 8-oxo-dG
DMPO Traps DNA Radicals and Prevents 8-oxo-dG DMPO Traps DNA Radicals and Prevents 8-oxo-dG Formation and DNA FragmentationFormation and DNA Fragmentation
Ramirez DC, 10 of 21
DN
A+
H2O
2
+ C
u(I
I)+
10
+ 1
00
+ 5
00
[DMPO] (mM)
Agarose gel electrophoresis
Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127
[BB] (mM)
Imm
un
ore
acti
vity
(R
LU
)
PB + 6.25 + 12.5 + 25.0 + 50.00
900
1800
2700
3600
4500
anti-DMPO anti-8-oxo-dG
† † †
**
**
COCO22 Enhances DNA Radicals, but Prevents 8-oxo- Enhances DNA Radicals, but Prevents 8-oxo-
dG and DNA Fragmentation by Copper-catalyzed dG and DNA Fragmentation by Copper-catalyzed OxidationsOxidations
BB: (bi)carbonateRamirez DC, 11 of
21Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127
Role of CORole of CO22 in Copper-catalyzed Damage to DNA in Copper-catalyzed Damage to DNA
DNA radicals
DNA-[Cu(I)O, Cu(II)/•OH or Cu(III)]
(Hydroxyl radical-like species)
DNA-[Cu(I)/(II)]
(DNA-bound copper)
DNA + Cu(II)
H2O2
H2O2
Catalase
or KCN
DTPA or BCDS
Oxidation and fragmentation
CO2 + HO2—
CO3• –
HOOCO2—
DNA-[Cu(I)] / HOOCO2—
DNA-[Cu(II)] + H2O
Oxidation, but not fragmentation
Ramirez DC, 12 of 21Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127
Detection of DNA Radicals in Functioning Cells by Immuno-spin Trapping
Some Applications
Ramirez DC, 13 of 21Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127
Why Has Spin Trapping Why Has Spin Trapping in vivoin vivo Been so Been so Challenging?Challenging?
DMPO (232 mg/100g b.wt.) or over 20 mM average concentration was not lethal. Both gross pathology and histopathological examination of the major organs were essentially negative with no obvious evidence of cellular damage being observed. Schaefer C.F., Janzen, E.G., West, M.S., Poyer, J.L., Kosanke, S.D.: Free Radical Bio. Med. 21: 427-436, 1996
Ramirez DC, 14 of 21
DN
A n
itro
ne
add
uct
s(R
LU
)
Lung Liver Intestine0
500
1000
1500 Added Recovered
DNA Nitrone Adducts are Stable to DNA Extraction DNA Nitrone Adducts are Stable to DNA Extraction Procedure Procedure
Known calf-thymus DNA nitrone adducts
are added to tissue homogenates(4mg proteins /ml)
DNA is extracted•Phenol
•Chlorophorm/Phenol/isoamyl alcohol
•Chloroform/isoamilic
• Ethanol precipitation
Calf-thymus DNA nitrone adducts are
recovered
Ramirez DC, 15 of 21
PB
H2O2 (mM)
10.50.10
25 mM BB/PB
Immuno-slot blot
Copper-catalyzed DNA Nitrone Adducts in Rat Copper-catalyzed DNA Nitrone Adducts in Rat HepatocytesHepatocytes
Ramirez, D.C.; Gomez-Mejiba, S.E. & Mason, R.P. Nature Methods, 2006. 3(2): 123-127Ramirez DC, 16 of
21
[Cu(II)] (M)
Imm
un
ore
act
ivit
y
0 1 5 10 25 500
1000
2000
3000anti-DMPO
anti-8-oxo-dG
Hepatocyte nuclei
[H2O2] (mM)
An
ti-D
MP
Oim
mu
no
rea
cti
vit
y (
RL
U)
0 0.1 0.5 10
500
1000 PB 25 mM BB/PB
Whole hepatocytes
DNA Nitrone Adducts Induced by Copper-mediated DNA Nitrone Adducts Induced by Copper-mediated Paraquat (PQ) Toxicity in Rats Paraquat (PQ) Toxicity in Rats
Cu(II) and/or PQ given by intragastric
intubation
DMPO (1 g/Kg) intraperitoneal
DMPO (1 g/Kg) intraperitoneal
Sacrifice and tissues are removed
DNA extraction and ELISA analysis
Based on: Kadiiska, M.B. & Mason, R.P. 2002. Spectrochemica Acta Part A, 58: 1227-1239 Ramirez DC, 17 of
21
DN
A n
itro
ne
add
uct
s(R
LU
)
Saline Cu(II) PQ PQ/Cu(II)0
500
1000
1500
IntestineLiverLung
Production and Detection of
Protein Nitrone Adducts
Ramirez DC, 18 of 21
1. Mason R. P. 2004. Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with Immuno-spin trapping. Free Radic. Biol. Med. 36: 1214-1223.
2. Ramirez, D.C., Gomez Mejiba, S.E. & Mason, R.P. 2005. Mechanism of Hydrogen Peroxide-induced Cu,Zn-superoxide dismutase-centered radical formation as explored by immuno-spin trapping: the role of copper- and carbonate radical anion-mediated oxidations. Free Radic. Biol. Med. 38: 201-214.
3. Ramirez, D.C. & Mason, R.P. 2005. Immuno-spin trapping: Detection of protein-centered radicals. In: Current Protocols in Toxicology, Suppl. 24, 17.7.1-17.7.18, John Wiley & Sons, Inc.
4. Ramirez, D.C, Gomez Mejiba, S.E. & Mason, R.P. 2005. Copper-catalyzed protein oxidation and its modulation by carbon dioxide. J. Biol. Chem. 280: 27402-27411.
5. Deterding, L.J., Ramirez, D.C., Dubin, J.R., Mason, R.P. & Tomer, K.B. 2004. Identification of free radicals on hemoglobin from self-peroxidation using mass spectrometry and immuno-spin trapping. J. Biol. Chem. 279: 11600-11607.
Suggested References on Immuno-spin Suggested References on Immuno-spin Trapping of Protein RadicalsTrapping of Protein Radicals
Ramirez DC, 19 of 21
ConclusionConclusionss1. ESR spin-trapping applications in biological research
has been limited
. expensive instrumentation
. specialized technicians
2. Immuno-spin trapping assays are heterogeneous
3. DMPO traps intermediate species
4. Oxygen is an spin trap and competes with DMPO
5. DMPO prevents final oxidation consequences
6. Nitrone adducts stability
7. Immuno-spin trapping is a simple, reliable, economic, and specific method to detect oxidatively generated radicals within isolated and cellular proteins and DNA.Ramirez DC, 20 of
21
Acknowledgments
Dr. Ronald P. Mason
M.S. Sandra E. Gomez Mejiba
M.S. Jean Corbett
Dr. Leesa Deterding
Thanks Free Radical Metabolites Workgroup for building day-after-day such an excellent environment to work doing science !!!
Ramirez DC, 21 of 21