Dario C. Ramirez [email protected] Free Radical Metabolites Workgroup LPC/NIEHS/NIH/DHHS Ramirez DC, 1 of 21 Immuno-spin

Dario C. Ramirez

[email protected]

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

mailto:[email protected]

http://www.niehs.nih.gov/

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


[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


[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


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


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


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


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 !!!


Documents

Dario C. Ramirez [email protected] Free Radical Metabolites Workgroup LPC/NIEHS/NIH/DHHS Ramirez DC, 1 of 21 Immuno-spin