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1
Overview of Oxidative Stress in Toxicology
Clementina Mesaros, Nathaniel Snyder, and Ian A. Blair Centers for Cancer Pharmacology and Excellence in Environmental Toxicology University of Pennsylvania Philadelphia, PA
2
Oxidative stress: background
• Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) and the ability of a cell to detoxify them.
• Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA.
• ROS (such as superoxide) can be beneficial by acting as cellular messengers in redox signaling or as a way to attack and kill pathogens
• Over-production of ROS and RNS can cause disruptions in normal mechanisms involved in cellular signaling
3
Exogenous and endogenous sources of oxidative stress
Exogenous sources
Oxygen γ-irrradiation UV-irradiation
Drugs Environmental chemicals
Food Tobacco smoke
Xenobiotics
Endogenous sources
Cells (e.g. mitochondria) Enzymes (e.g. NO synt., CYP2E1) Metabolism (e.g. mitochondria)
Diseases (e.g. ischemia)
Reactive oxygen species (ROS) and/or Reactive nitrogen species (RNS)
Oxidative stress
4
Consequences of ROS and RNS generation
Kohen R, Nyska A. Toxicol Pathol. 2002;30:620
5
Electrophilic aldehydes and oxidative stress
Blair IA. J Biol Chem. 2008;283:15545
OH + Polyunsaturated fatty acids Lipid hydroperoxides
Homolytic decomposition
(ROS)
.
OC5H11
H
OOHO
C5H11H
OHO
C5H11H
O
+
4-hydroperoxy-2(E)-nonenal(HPNE)
4-hydroxy-2(E)-nonenal(HNE)
4-oxo-2(E)-nonenal(ONE)
6
15-Lipoxygenase (LOX)-1 transfected cell (R15LO)
Western blot for 15-LOX-1
Model system to study 15-LOX-1-mediated lipid peroxidation to 15(S)-HPETE
Rmock R15LO
Western blot for COX-2
Rmock R15LO
7
Oxidative stress and glutathione (GSH)
• Intracellular GSH provides a major defense against oxidative stress
• During oxidative stress reduced GSH is converted to glutathione disulfide (GSSG)
• The high abundance of GSH and low abundance of GSSG helps to maintain cells in a reducing environment helps to prevents oxidative damage to cellular macromolecules.
• Hydrogen peroxide and lipid hydroperoxides (eg 15-HPETE) undergo GSH peroxidase-mediated reduction to water or lipid hydroxides, respectively
8
Oxidation of glutathione and oxidative stress
Zhu P, Oe, T, and Blair IA. Rapid Commun Mass Spectrom . 2008;30:620
R15LO = Monocyte/macrophage cells transfected with 15-lipoxygensase gene RMock = Monocyte/macrophage cells transfected with empty plasmid
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GSH homeostasis and cellular redox status
• GSH forms adducts with a great variety of both endogenous and exogenous reactive electrophilic intermediates, generally facilitated by GSH S-transferases (GSTs)
• This is considered to normally represent a detoxification of the relevant reactive intermediate
• Changes of the half-cell reduction potential of the 2GSH/GSSG couple correlate with the biological status of the cell
• GSH/GSSG homeostasis plays an important role in maintaining cellular redox status. Determinations of the reduction potential (Nernst equation) can be used to monitor oxidative stress.
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Redox status determined from GSSG/GSH ratio
Expression of 15(S)-hydroperoxy-eicosatetraenoic acid up-regulated anti-oxidant defense
Zhu P, Oe, T, and Blair IA. Rapid Commun Mass Spectrom . 2008;30:620
11
Nrf2 mediated antioxidant response
Binding of electrophiles such as ONE to Keap1 SH-groups releases Nrf2, which up-regulates GSH biosynthesis and other anti-oxidant responses
J Exp Biol 2006;209:2337
12
Biomarkers of oxidative stress: background
Type Biomarker Reference
DNA 8-oxo-2'-deoxyguanosine (8-oxo-dGuo) Mesaros et al. Free Radic Biol Med. 2012;53:610
DNA 8-oxo-2'-deoxyguanosine (8-oxo-dAdo) Cooke et al. FASEB J. 2003;17:1195
DNA Cyclo-dA and Cyclo-dG Yuan Nucleic Acids Res. 2011;14:5945
DNA Etheno DNA-aducts (etheno-Ade) Chen and Kao Tox Lett. 2007;169:72
DNA Malondialdehyde dGuo adduct (M1G) Jeong at al. Chem Res Tox. 2005;18:51
lipid Malondialdehyde (MDA) Lee et al. Curr Med Chem. 2012;19:2504
lipid Isoprostanes Milne at al. Chem Rev. 2011;111:5973
lipid Acrolein Wang et al. Biomarkers 2011;16:144
protein Nitrotyrosine Tsikas D. Amino Acids. 2012;42:45
13
Urinary 8-oxo-dGuo: non-smokers vs smokers
0
2
4
6
8
* p<0.05
Non-Smokers Smokersn=48 n=84
(A)
Subjects
8-ox
o-dG
uo(n
g/m
L)
0
1
2
3
Non-Smokers Smokersn=48 n=84
* p<0.05
(B)
Subjects8-
oxo-
dGuo
(nm
ol/m
mol
cre
atin
ine)
Mesaros et al. Free Radic Biol Med. 2012;53:610
Normalized to creatinine Concentration (ng/mL)
14
Asbestos-induced oxidative stress
ROS
NF-kBinflammasome
NLRP3
IL-1βIL-18
NF-kB
Lipids
8-oxo-dGuo TNFα
TNFα
MDAIsoprostanes
Modified lipidsAsbestos
Transcription
serum biomarkers
cytosol
nucleus
mesothelialcell
membrane
180 nm
5 µm
15
Asbestos-induced formation of lung 8-oxo-dGuo 8-
oxo-
dGuo
in lu
ng/1
06 d
Guo
Control Crocidolite asbestos Forsterite (inactivated crocidolite)
Days after intratracheal administration
Takata A. et al Inhal. Toxic. 2009;9:739
16
Formation of urinary etheno-adenine
OH + Polyunsaturated fatty acids Lipid hydroperoxides
Homolytic decomposition
OC5H11
H
OOHN
NN
NO
HO
OH
N
NH
NN
N
NdAdo
4-hydroperoxy- 2(E)-nonenal etheno-dAdo etheno-Ade
(ROS)
Lee et al. Chem Res Toxicol. 2005;18:780
.
17
Urinary etheno-Ade: non-smokers vs smokers
Chen HJ, Kao CF. Tox Lett. 2007;169:72
Normalized to creatinine Concentration (pg/mL)
0
20
40
60
80
100
Subjects
Eth
eno-
Ade
(pg/
mL)
* p < 0.0004
Non-smokers(n=10)
Smokers(n=18)
0
20
40
60
80
100
SubjectsE
then
o-A
de(p
mol
/nm
ol c
reat
inin
e)
* p < 0.003
Non-smokers(n=10)
Smokers(n=18)
18
Formation of urinary heptanone-etheno-dGuo
DNA
vinyl chloride, vinyl f luoride,chloracetaldehyde
OOC5H11
H -H2O
ONE
OOOHC5H11
H
N
HOCH2
OH
NN
ONH
NN N
N
HOCH2
OH
O
15(S)-HPETE
DNA
HPNE
etheno-dGuo etheno-dCyd
NH
NN N
N
HOCH2
OH
OO
C5H11
N
HOCH2
OH
NN
O
O
C5H11DNA
heptanone-etheno-dGuo(HεdGuo)
heptanone-etheno-dCyd(HεdCyd)
Pollack M, Yang IY, Kim HY, Blair IA, Moriya M. Chem Res Toxicol. 2006;19:1074
19
Urinary heptanone-etheno-dGuo (HεdGuo)
NS Smoker0
10
20
30
40
50
60
*Hε d
Guo
(ng/
mL)
20
Isoprostane formation during oxidative stress
Milne et al. Chem Rev. 2011;111:5973
CO2H
CO2HOO
CO2HOH
CO2H
CO2H
HO
HO
5
8-series F2-isoprostanes
OH/O2
5-series F2-isoprostanes
O OCO2H
CO2HHO
HO OH
12
12-series F2-isoprostanes
OO
OHCO2H
8
O2
O2O2
OO
HO
HO15-series F2-isoprostanes
O2
OH
CO2H15
HO
HO
OH/O2
arachidonic acid
21
Manganese-induced isoprostane formation
Milatovic et al. Toxicol Appl Pharmacol. 2009;240:219
Cerebral F2-isoprostane formation in mice
22
Urinary 8-iso-PGF2α: non-smokers vs. smokers
23
Urinary 8-iso-PGF2α: relationship with nicotine
Free 8-iso-PGF2α Total 8-iso-PGF2α
24
20-HETE levels in urine in smoker vs. non-smokers
Non Smoker0
200
400
600
800
1000NonSmoker
p=0.04
smoking status
20-H
ETE
(pg/
g cr
eatin
ine)
25
Formation of nitrotyrosine during oxidative stress
Tsikis D. Amino Acids. 2012;42:45
26
Nitrotyrosine formation during reperfusion injury
Liu et al. Biochim Biophys Acta. 2009;1794:476
Nitrated peptide from mitochondrial complex I sub-unit isolated from mouse heart
27
Nitrated fibrinogen in venous thromboembolism
Martinez et al. Free Radic Biol Med. 2012;53:230
(n= 178) (n= 48)
VTE = venous thromboembolism
28
Oxidative stress symposium
1:45 pm Quantitative analysis of thiol electrophile and redox modifications by LC-MS/MS Daniel C Liebler, Jing Yang, Keri A Tallman, Vinayak Gupta, Kate S Carroll, Ned A Porter
2:25 pm HDL oxidation in the pathogenesis of cardiovascular disease
Baohai Shao, Jay W Heinecke 3:05 pm Intermission 3:20 pm Cyclo-dA and Cyclo-dG as biomarkers of oxidative stress
Yinsheng Wang 4:00 pm Antimalarial drug artesunate ameliorates oxidative lung
damage in experimental allergic asthma W E Ho, C Cheng, H Y Peh, F Xu, C N Ong, W S Wong, J. S. Wishnok, Ravindra Kodihalli, Steven R Tannenbaum
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