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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

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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

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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

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Consequences of ROS and RNS generation

Kohen R, Nyska A. Toxicol Pathol. 2002;30:620

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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)

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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

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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

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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

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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

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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

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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)

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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

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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

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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

.

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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)

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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

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Urinary heptanone-etheno-dGuo (HεdGuo)

NS Smoker0

10

20

30

40

50

60

*Hε d

Guo

(ng/

mL)

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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

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12-series F2-isoprostanes

OO

OHCO2H

8

O2

O2O2

OO

HO

HO15-series F2-isoprostanes

O2

OH

CO2H15

HO

HO

OH/O2

arachidonic acid

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Manganese-induced isoprostane formation

Milatovic et al. Toxicol Appl Pharmacol. 2009;240:219

Cerebral F2-isoprostane formation in mice

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Urinary 8-iso-PGF2α: non-smokers vs. smokers

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Urinary 8-iso-PGF2α: relationship with nicotine

Free 8-iso-PGF2α Total 8-iso-PGF2α

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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)

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Formation of nitrotyrosine during oxidative stress

Tsikis D. Amino Acids. 2012;42:45

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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

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Nitrated fibrinogen in venous thromboembolism

Martinez et al. Free Radic Biol Med. 2012;53:230

(n= 178) (n= 48)

VTE = venous thromboembolism

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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