Larry Marnett Cellular Responses to Aldehydic Products of Lipid Peroxidation.
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Transcript of Larry Marnett Cellular Responses to Aldehydic Products of Lipid Peroxidation.
![Page 1: Larry Marnett Cellular Responses to Aldehydic Products of Lipid Peroxidation.](https://reader036.fdocuments.in/reader036/viewer/2022062519/56649d2f5503460f94a07e55/html5/thumbnails/1.jpg)
Larry MarnettCellular Responses to Aldehydic Products of Lipid
Peroxidation
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NOO2–
H2O2
HO•
NO
O–O
hydroperoxides, endoperoxides,isoprostanes
MDA, HNE, ONE
NN
N NN
O
gene expression
apoptosis
replication blockmutationdamage signaling
degradation
Proteins
oxidativemetabolism
GSHconjugation
PLA2
hydrolysis
Fe2+ or
ascorbate
p53cell cycle arrest
reduction
+
Aldehyde Mediators of Oxidative DamageAldehyde Mediators of Oxidative Damage
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R C5H11Lip. Perox.
O2
R C5H11
H
OC5H11
OOH
H
OC5H11
OH
H
O OH
HPNE
ONE HNE
OOH
H
O O
H
H
OC5H11
O
MDA
Diffusible Electrophiles from Lipid PeroxidationDiffusible Electrophiles from Lipid Peroxidation
Ox
Red-H2O
Ox
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Lipid Peroxidation-Induced DNA DamageLipid Peroxidation-Induced DNA Damage
H
OC5H11
OH
HN
N N
N
O
H2N
N
N N
N
O
NH
OH
HO
C5H11
H
OC5H11
O
N
N N
N
NH2
N
N N
N
NH
O
H11C5
+
+
H-εdA
OH-Hexyl-PdG
O
H
OH
HN
N N
N
O
H2N
N
N N
N
O
N
M1dG
+
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N
N N
N
NH
O
H11C5
H-εdA
N
N N
N
O
NH
OH
HO
C5H11
OH-Hexyl-PdG
N
N N
N
O
N
M1dG
M1dGTM1dGAframeshifts
OH-Hexyl-dGTOH-Hexyl- dGA
Mutagenic Spectrum of Lipid Peroxidation-Induced AdductsMutagenic Spectrum of Lipid Peroxidation-Induced Adducts
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M1dGTM1dGdAframeshifts
OH-Hexyl-dGTOH-Hexyl- dGdA
N
N N
N
NH
εdA
N
N N
N
O
NH
OH
HO
C5H11
OH-Hexyl-PdG
N
N N
N
O
N
M1dG
εdA dG
Mutagenic Spectrum of Lipid Peroxidation-Induced AdductsMutagenic Spectrum of Lipid Peroxidation-Induced Adducts
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HNE and ONE Protein AdductsHNE and ONE Protein Adducts
CRT,8, 284, (1995); CRT, 16, 1598 (2003)
plus unstable Michael adducts
ONE is 100x more reactive than HNE
H
OC5H11
OH
OHO
XR
R = Cys, His, Lys
OHN
XR
Lys
N
C5H11 C5H11
C5H11
LysH
NC5H11
OH
Lys
Cys, His, Lys
Lys
Lys –H2O
H
OC5H11
O
ArgN
N
H2N
NH3+
CO2H
ONE
HNE
C5H11O
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Enedials from Other SourcesEnedials from Other Sources
H
OC5H11
O
ONE
P-450
O2 OO
HO
OH
OO O
O
O
R
P-450
O2
HO O
H
R
OH
ROH
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HNE Induction of Apoptosis and NecrosisHNE Induction of Apoptosis and Necrosis
CRT, 14, 1090 (2001)
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H
OC5H11
OOH
H
OC5H11
OH
H
OC5H11
O
H
OC5H11
OH
HPNE ONE (R)-HNE (S)-HNE
Toxicity of Peroxidation-Derived AldehydesToxicity of Peroxidation-Derived Aldehydes
CRT, 17, 453 (2004)
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Depletion of GSH by HNEDepletion of GSH by HNE
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Apoptosis Induced by HPNE and ONE in RKO CellsApoptosis Induced by HPNE and ONE in RKO Cells
CRT, 17, 453 (2004)
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membranemembranereceptor?receptor?
Cas-3Cas-3
Procas-3Procas-3 Cas-8Cas-8
Cell membraneCell membrane
MitochondriaMitochondriaBcl-2Bcl-2
Cas-9Cas-9
Apaf-1Apaf-1
Cyt cCyt c
H
O
OH
4-HNE
PARP-fodrin
DFF45/35
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Protein Synthesis is Required forProtein Synthesis is Required forHNE-Mediated ApoptosisHNE-Mediated Apoptosis
What transcriptional pathways are altered by HNE?What transcriptional pathways are altered by HNE?
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Reduced Apoptosis in p53Reduced Apoptosis in p53-/--/- Cells Exposed to HNE Cells Exposed to HNE
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NFNFB SignalingB Signaling
Li et al, Mol.Immunol., 41, 701 (2004)
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. .
/TPA IM (30 )- /HNE ' TPA IM
0 5 15 30 0 5 15 30
Supershift
-NF B
1 2 3 4 5 6 7 8 9 10 11
( )min
JBC, 276, 18223 (2001)
HNE Inhibits NFHNE Inhibits NFB Translocation and DNA BindingB Translocation and DNA Binding
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HNE Inhibits NFHNE Inhibits NFB-Dependent TranscriptionB-Dependent Transcription
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.
32P-IB-GST ( )IKK activity
( )HNE µM - - - 30 60
Samples
1 2 3 4 5
IKK
-GST IB
220 K
97 K
220 K
97 K
HNE modified IKK ( -IB with anti IKK)
HNE modified IKK ( - )IB with anti HNE
HNE Modification of IHNE Modification of IB KinaseB Kinase
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SH HSSH HS
Structure and Regulation of IStructure and Regulation of IB KinaseB Kinase
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NOO2–
H2O2
HO•
NO
O–O
hydroperoxides, endoperoxides,isoprostanes
MDA, HNE, ONE
NN
N NN
O
gene expression
apoptosis
replication blockmutationdamage signaling
degradation
Proteins
oxidativemetabolism
GSHconjugation
PLA2
hydrolysis
Fe2+ or
ascorbate
p53cell cycle arrest
reduction
+
Oxidative Stress and Its ConsequencesOxidative Stress and Its Consequences
IK
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Doses
5, 20, and 60 µM HNE
Array Type
AffymetrixAffymetrix
U133 Plus 2.0 Chip~55,000 Probes
~47,000 Transcripts
Times
6 and 24 h
Microarray Experiments:Dose Response, Time Course, and Chip Specifications
CRT, 18, 1641 (2005)
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4 22
115
0
0 11
3
5 µM HNE 20 µM HNE
60 µM HNE
54520
all genes
Genes Up-regulated at 6 h Using MicroarrayGenes Up-regulated at 6 h Using Microarray
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2 742
20 µM HNE 60 µM HNE
54597
all genes
Genes Up-regulated by HNE at 24 hGenes Up-regulated by HNE at 24 h
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6 h 24 hiological Response/Gene Acc. No. 20 µM HNE 60 µM HNE 60 µM HNEntioxidant Responseeme oxygenase 1 (HMOX1) NM_002133 8.4 9.1 7.4utamate-cysteine ligase, modifier subunit (GCLM)† NM_002061 3.4 3.1 –AD(P)H-dependent quinone oxidoreductase (NQO1)† NM_000903 2.5 – –lute carrier family 7, member 11 (SLC7A11, xCT)† NM_014331 8.7 6.1 –ioredoxin reductase 1 (TXNRD1) NM_003330 2.4 9.3 5.9
eat Shock ResponsenaJ homolog, subfamily A, member 4 (DnaJA4)† NM_018602 –§ 4.4 3.4naJ homolog, subfamily B, member 1 (DnaJB1)† NM_006145 – 4.3 4.4naJ homolog, subfamily B, member 2 (DnaJB2) NM_006736 – 4.6 3.6naJ homolog, subfamily B, member 4 (DnaJB4)† NM_007037 – 5.7 2.1eat shock 70kDa protein 1A (HSPA1A) NM_005345 – 3.0 3.6eat shock 110kDa protein family (APG-1) NM_014278 – 2.6 –eat shock 105/110kDa protein 1 (HSPH1)† NM_006644 – 9.6 12.1
R Stress/Amino Acid/Nutrient Sensing Responseomocysteine, ER stress-inducible protein 1 (HERP) NM_014685 – 4.9 –tivating transcription factor 3 (ATF3) NM_001674 – 5.1 –lute carrier family 3, member 2 (SLC3A2) NM_002394 – 2.7 –paragine synthetase (ASNS) NM_001673 – 2.8 –
ystathionase (CTH)† NM_001902 – 3.9 –owth arrest, DNA damage-inducible 34 (Gadd34)† NM_014330 – 3.4 3.2bbles homolog 3 (TRB3)† NM_021158 – 3.3 –
ell Signalingsulin-induced gene 1 (INSIG1)† NM_005542 – 2.3 –AP kinase kinase kinase 8 (MAP3K8) NM_005204 – 3.5 2.0DC-like kinase 1 (Clk1) NM_004071 – 3.8 3.2acental growth factor (PGF) NM_002632 – 3.5 4.9
onnective tissue growth factor (CTGF) NM_001901 5.2 4.5 3.5gnal-induced proliferation-associated 1-like 2 (SIPA1L2)† NM_020808 3.8 3.6 –drenomedullin (ADM) NM_001124 – 3.5 4.0TP binding protein expressed in skeletal muscle (GEM) NM_005261 – 4.9 5.5belson leukemia viral oncogene homolog 2 (ABL2) NM_005158 – 3.0 –broblast growth factor receptor substrate 2 (FRS2) NM_006654 – – 2.3AS-like without CAAX1 (RIT1) NM_006912 – 2.6 –
poptotic Regulationowth arrest, DNA damage-inducible 45β (Gadd45β)† NM_015675 – 5.6 3.8usterin (Clus)† NM_001831 – 3.2 3.9urine double minute 2, hum anhomolo g(HDM2)† NM_002392 – – 2.5
ell Cycle Regulationstrin 2 (SESN2)† NM_031459 – 5.4 –yclin G2 (CCNG2)† NM_004354 3.2 2.8 –gulato -r of Gprotein signalin g2 (RGS) NM_002923 – 3.7 3.7ycli -n depende ntkina -selike kina 3 (se CDKL3) NM_016508 – 2.6 –urine-rich bindin g element protein (A PURA) NM_005859 – 3.2 3.7SP -Y lik e2 (TSPYL2)IO kinase 3 (RIO 3K ) NM_003831 – – 2.4
ip id Metabolis man dTr ansportytochro meP450 39A1 (CYP39 1A ) NM_016593 6.1 7.3 –xystero l bindin g protei -n lik 1 (e AOSBPL1A) NM_018030 – 2.3 –
NA Processin /gNuclea r Transportolyadenylatio n element binding protein 3 (CPEB3) NM_014912 – 4.3 –ownregulate din ovaria ncan cer1 (DO 1C ) NM_014890 – 3.2 –eath effector domain, -DNA binding domain 2 (DEDD2) NM_133328 – 3.0 2.2olyadenylatio n element binding protein 4 (CPEB4)† NM_030627 – 3.0 2.6ucleoporin like 1 (NUPL1) NM_014089 – – 2.1un -o like 6, RNA binding prote in(BRUNOL6) NM_052840 – – 3.3
ytoskeletal Function/Structural Maintenanceocollagen-proline 4-hydroxylase (P4HA2)† NM_004199 – 3.3 –tin related protein M1 (ARPM1) NM_032487 – 9.2 4.1ldesmon 1 (CALD1) NM_004342 2.4 – –
ansgelin (TAGLN) NM_003186 2.3 – –rine protease inhibitor H1 (SERPINH1) NM_001235 – – 2.6
ranscriptional Regulationdult retina protein (LOC153222)† NM_153607 – 2.1 –rly growth response 1 (EGR1) NM_001964 – 2.6 3.2nc finger and BTB containing protein 20 (ZBTB20) NM_013360 – 2.9 3.5-box protein 3 (TBX3) NM_005996 – 3.1 –MB-box transcription factor 1 (HBP1) NM_012257 – 2.7 –nc finger protein 10 (ZNF10) NM_015394 – 3.2 –nc finger protein 227 (ZNF227) NM_182490 – 2.5 –nc finger protein 267 (ZNF267) NM_003414 – 2.2 –
AMP response element binding protein 3-like 4 (CREBP3L4) NM_130898 – 2.6 –AX dimerization protein 1 (MAD) NM_002357 – 3.1 –nc finger protein 554 (ZNF554) NM_152303 – 2.4 2.3maf oncogene homolog f (MAFF)† NM_012323 – 3.7 –nc finger protein 12 (ZNF12) NM_033204 – – 2.2ING1 and YY1 binding protein (RYBP) NM_012234 – – 2.5rin (PIR) NM_003662 2.5 – 2.5ADS box transcription enhancer factor 2A (MEF2A) NM_005587 – – 2.2nc finger protein 277 (ZNF277) NM_021994 – – 3.1nc finger protein 79 (ZNF79) NM_007135 – – 3.3nc finger protein 556 (ZNF556) NM_024967 – – 4.2monji domain containing 1C (JMJD1C) NM_004241 – 2.4 –ixed lineage leukemia-trithorax homolog 2 (MLLT2) NM_005935 – 2.9 –x-determining region Y-b ox 18 (SOX18) NM_018419 – – 21.8
n Homeostasis and Cellular Transporta2+-transporting ATPase, type 2C, member 1 (ATP2C1) NM_014382 – 2.9 –holinergic receptor, nicotinic, alpha polypeptide 5 (CHRNA5) NM_000745 – 2.7 –daptor-related protein complex 4, β1 subun it (AP4β1) NM_006594 – 2.2 –ucleosid e diphosphat -e linked moie ty -X typ 9 (e NUDT9) NM_024047 – 2.4 –lut e carrie r family 16, member 14 NM_152527 2.4 – –
NA Repairbiquitin-conjugatin g enzym eE2 B (RAD6 homolo ) (g UBE2B) NM_003337 – 4.0 –RC -A 1-interacting prote in C-terminal helica 1 (se BRIP1) NM_032043 2.3 – –
istonesstone 1, H3 (c HIST1 3H E) NM_003532 – 2.7 –stone 1, H2b (k HIST1 2H BK) NM_080593 – – 2.5stone 1, H2b (g HIST1 2H BG) NM_003518 – – 9.6stone 2, H2a a(HIST2 2H AA) NM_003516 – – 2.6
rotein Tr anslation/Amino Acid Metabolismukaryoti c translatio ninitiatio nfactor 5 (EIF5)† NM_001969 – – 2.5itochondria l ribosomal protein L18 (MRPL18) NM_014161 – 2.2 –oleucin -e tRNA synthetase (IARS) NM_002161 – 4.9 3.5hydrofolate reduct -aselik 1 (e DHFRL1) NM_176815 3.5 – –rosina -se relat edprotein 1 (TYRP1) NM_000550 3.7 – –
6 h 24 hiological Response/Gene Acc. No. 20 µM HNE 60 µM HNE 60 µM HNE
Known Genes Upregulated by HNEKnown Genes Upregulated by HNE
HNE Stimulates Many DifferentHNE Stimulates Many DifferentTranscriptional PathwaysTranscriptional Pathways
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HNE Induces Gene Expression via Many Signaling PathwaysHNE Induces Gene Expression via Many Signaling Pathways
ResponseTranscription
FactorsInduced Genes
AntioxidantResponse
Nrf2 HMOX1, NQO1, GCLM, Xc–
Misfolded/DamagedProtein Response;
Amino Acid Starvation
HSFs, ATF4,ATF6, XBP1
DnaJA4, HspA6, Hsp70B,Gadd34, ASNS
DNA DamageResponse
p53 Mdm2, cyclin G2, p21
General Stress/Unknown
Other/Unknown Gadd45β, Clusterin, CYP39A1
Stress-ActivatedKinase Signaling
AP-1 stress-responsive genes
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Induction of ARE Target Genes by HNEInduction of ARE Target Genes by HNE(Real Time RT-PCR)(Real Time RT-PCR)
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Induction of CYP39A1 by HNEInduction of CYP39A1 by HNE
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Increase in Protein Levels Following HNE TreatmentIncrease in Protein Levels Following HNE Treatment
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Activation of Stress-ResponsiveActivation of Stress-ResponsiveTranscription Factors by HNETranscription Factors by HNE
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Activation of NRF2Activation of NRF2
CRT, 18, 1779 (2005)
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Knock-down of Nrf2 by siRNA
Nrf2 siRNA transfection
0
2
4
6
8
10
12
0 MG132, 5 uM 0 MG132, 5 uM 0 MG132, 5 uM
post-transfection treatment
fmol per ug RNA
(x10
-5)
83% 82%
percent reduction (to neg control siRNA)
NO transfection Negative Control siRNA Nrf2 siRNA
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β-tubulin (50 kDa)
Nrf2 (68 kDa)
siRNA
MG-132 (5 M)
none none control control Nrf2 Nrf2
- + - + - +
nuclear protein blot
Knock-down of Nrf2 by siRNA
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Knock-down of Nrf2 by siRNA
HNE-treatment
HO-1 mRNA (effect of Nrf2 knock-down)
0
5
10
15
20
25
30
0 HNE 15uM HNE 45uM 0 HNE 15uM HNE 45uM 0 HNE 15uM HNE 45uM
post-transfection treatment
fmol per ug RNA
(x10
-4)
NO transfection Negative Control siRNA Nrf2 siRNA
45%
46%
64%
percent expression(to neg control siRNA)
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Activation of Heat-Shock Signaling by Hsp90 ModificationActivation of Heat-Shock Signaling by Hsp90 Modification
CRT, 19, 173 (2006)
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JPET, 315, 8 (2005); Chem.Res.Toxicol.18, 1324 (2005)
Immunochemical Detection of HNE-Adducted ProteinsImmunochemical Detection of HNE-Adducted Proteinsin Alcoholic Liver Diseasein Alcoholic Liver Disease
Microsomal Mitochondrial
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HN NH
S
HN
O
O
O
HN
O
PPh2
O
CH3O
3
Protein N3 +HN NH
S
HN
O
O
O
HN
O
PPh2
NH
O
3
Protein
O
O
H
OH
N3
Protein Labeling with Azido-HNEProtein Labeling with Azido-HNE
Aaron Jacobs
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Analysis of Proteins Modified in RKO cellsAnalysis of Proteins Modified in RKO cells
0 M 100 M50 M-FBS+FBS -FBS +FBS
Results:Protein labeling more prominent in cells incubated with media lacking FBS
Most labeled proteins are high Mol. Wt.
Protein: 5 g/lane4-20% SDS-PAGE gradient gelNitrocellulose (0.2 m)Probe with S/A-HRP
Std
Andy Vila
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Putative Targets of Electrophilic Lipid Peroxidation Products
HPNE, ONE, HNE
gene expression
GSH
enhanced oxidantsensitivity
IB Kinase
sensitivity to apoptosis
Protein Phosphatases
DNA
Keap1
antioxidantresponse
mutationsdamagesignaling
enhanced kinaseactivation
Ca2+-ATPase
ER Stress
Low level stress ProtectionHigh level stress Apoptosis
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Not All Aldehydes Are Created EqualNot All Aldehydes Are Created Equal
H
OC5H11
OH
HNEExtensive changesin gene expression
and signalingp53-Dependent-Apoptosis
H
O OH
MDAChanges
in gene expressionand signaling
p53-DependentG1/S and G2/M block
No Apoptosis
H
O OH
R
-Substituted-MDAR = alkyl/aryl
No signaling changesNo Apoptosis/No Necrosis
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H
O OH
N
H
O OH
N
N
Induction of p53-Dependent Cell Cycle Arrest andInduction of p53-Dependent Cell Cycle Arrest andApoptosis by QD-MDAApoptosis by QD-MDA
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Aldehyde Mediators of Oxidative DamageAldehyde Mediators of Oxidative Damage
NOO2–
H2O2
HO•
NO
O–O
hydroperoxides, endoperoxides,isoprostanes
MDA, HNE, ONE
Fe2+ orascorbate
reduction
+
(DIFFUSIBLE ELECTROPHILES)
O
O
OP
O
O
O
NHCl
H31C15
O
(CH2)3CO2H
O
O
O
OP
O
O
O
NH31C15
O
O
O
H
PGPC POPC
O
O
OP
O
O
O
NH31C15
O
O
OH
H
O
HOOA-PC
O
O
OP
O
O
O
NH31C15
O
O
OO
OH
C5H11
PEIPC
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O
O
OP
O
O
O
NH31C15
O
O
O
H
Induces apoptosis in A549 cells at 100-200 nM
Uhlson et al, Chem.Res.Toxicol. 15, 896, 2002
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Aldehyde Mediators of Oxidative DamageAldehyde Mediators of Oxidative Damage
NOO2–
H2O2
HO•
NO
O–O
hydroperoxides, endoperoxides,isoprostanes
MDA, HNE, ONE
Fe2+ orascorbate
reduction
+
(DIFFUSIBLE ELECTROPHILES)
O
O
OP
O
O
O
NHCl
H31C15
O
(CH2)3CO2H
O
O
O
OP
O
O
O
NH31C15
O
O
O
H
PGPC POPC
O
O
OP
O
O
O
NH31C15
O
O
OH
H
O
HOOA-PC
O
O
OP
O
O
O
NH31C15
O
O
OO
OH
C5H11
PEIPC
Protein Targets, Biological Effects?Protein Targets, Biological Effects?
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Chuan Ji
JamesWest
AaronJacobs
Andy Vila
CRT, 19, 173 (2006)
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Carmelo Rizzo
Alan Brash
Ned Porter
Jianxin Ji
National Foundation for Cancer Research
National Institutes of Health
AcknowledgementsAcknowledgements
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Sowell et al, PNAS, 101, 17964 (2004)
1.3 µM inhuman plasma
HNE-Ascorbate Conjugate Found In VivoHNE-Ascorbate Conjugate Found In Vivo