Oxidative stress Oxidative Stress Reactive oxygen species ROS

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Oxidative stress

Oxidative stress

Oxidative Stress • • Reactive oxygen species (ROS) ROS and oxidative stress Antioxidant system

Oxidative Stress • • Reactive oxygen species (ROS) ROS and oxidative stress Antioxidant system Oxidative damage Oxidative stress and apoptosis Oxidative stress and aging Oxidative stress and cancer ROS as signaling molecules

Reactive oxygen species (ROS) • ROS – OH. (hyroxyl radical) – O 2 -.

Reactive oxygen species (ROS) • ROS – OH. (hyroxyl radical) – O 2 -. (superoxide radical) – H 2 O 2 (hydrogen peroxide) – NO. (nitric oxide) • Oxidative stress • Oxidative damage

Toxic effects of ROS • Protein oxidation • Lipid peroxidation • Nucleic acids damage

Toxic effects of ROS • Protein oxidation • Lipid peroxidation • Nucleic acids damage – Double-strand DNA breaks – Single-strand DNA breaks – Change DNA bases • 8 -oxoguanine • Thymine glycol

Lipid peroxidation • Measure the malondialdehyde formed • Lipid peroxidation is a chain reaction.

Lipid peroxidation • Measure the malondialdehyde formed • Lipid peroxidation is a chain reaction. • Each fatty acyl moiety that undergoes peroxidaion generate a radical that can initiate another peroxidation reaction.

Intracellular sources of free radicals • Mitochondrial electron transport system – Superoxide radical and

Intracellular sources of free radicals • Mitochondrial electron transport system – Superoxide radical and semiquinone radical • Microsomal (ER) electron transport system – Superoxide radical and H 2 O 2 • Arachidonic acid metabolism • Reactions within peroxisome – Superoxide radical and H 2 O 2

 • H 2 O 2 and O 2 -. may diffuse from their

• H 2 O 2 and O 2 -. may diffuse from their subcellular sites of production and affect the whole cell • H 2 O 2 can cross biological membranes

NO. • Small • Gas • Synthesize by nitric oxide synthase (NOS) – n.

NO. • Small • Gas • Synthesize by nitric oxide synthase (NOS) – n. NOS: constitutive expression – e. NOS: constitutive expression – i. NOS: inducible

Reactive nitrogen species (RNS) • Inactivation of respiratory chain complexes; inhibition of protein and

Reactive nitrogen species (RNS) • Inactivation of respiratory chain complexes; inhibition of protein and DNA synthesis • RNS are reduced or inactivated through the generation of a disulfur bond between two glutathione molecules to form oxidized glutathione

Antioxidative system • Antioxidant – Glutathione, GSH – Vitamin C, E – Cysteine –

Antioxidative system • Antioxidant – Glutathione, GSH – Vitamin C, E – Cysteine – Protein-thiol – Cerutoplasmin: important in reducing Fe 3+ release from ferritin • Antioxidative enzyme

Glutathione (GSH)

Glutathione (GSH)

Antioxidative enzyme • • Catalase Superoxide dismutase Glutathione peroxidase Glutathione reductase Gluththione S-transferase Glucose-6

Antioxidative enzyme • • Catalase Superoxide dismutase Glutathione peroxidase Glutathione reductase Gluththione S-transferase Glucose-6 -phosphate dehydrogenase DT-diaphorase

Oxidants as stimulators of signal transduction • Oxidants – Superoxide – Hydrogen peroxide –

Oxidants as stimulators of signal transduction • Oxidants – Superoxide – Hydrogen peroxide – Hydroxyl radicals – Lipid hydroperoxides

ROS act as second messengers • Ligand-receptor interactions produce ROS and that antioxidants block

ROS act as second messengers • Ligand-receptor interactions produce ROS and that antioxidants block receptormediated signal transduction led to a proposal that ROS may be second messengers

Reactive oxygen species (ROS) as second messengers • Generation of ROS by cytokines Ligand

Reactive oxygen species (ROS) as second messengers • Generation of ROS by cytokines Ligand Tumor necrosis factor- Interleukin 1 Transforming growth Factor- 1 Platelet derived growth factor Insulin Angiotension II Vitamin D 3 Parathyroid hormone ROS H 2 O 2/HO H 2 O 2/O 2 - H 2 O 2/O 2 -

 • TNF activates oxidative stress-responsive transcription factors, NF- B and AP-1, and also

• TNF activates oxidative stress-responsive transcription factors, NF- B and AP-1, and also induces apoptosis.

ROS induce apoptosis • Both H 2 O 2 and menadione induce neuronal cells

ROS induce apoptosis • Both H 2 O 2 and menadione induce neuronal cells apoptosis. • Decreased superoxide dismutase activity was found to cause apoptosis in neuronal cells • Apoptosis induced by HIV infection was inhibited by antioxidant such as Nacetylcysteine, catalase, vitamin E, and 2 mercaptoethanol

ROS induce apoptosis • Bcl-2 protects cells from TNF -induced apoptosis in mouse L

ROS induce apoptosis • Bcl-2 protects cells from TNF -induced apoptosis in mouse L cells. • Bcl-2 was ineffective in influencing TNF signaling for NF- B activation in these cells. • H 2 O 2 activates the DNA binding activity of p 53. P 53 is required for the induction of apoptosis.

ROS measurement

ROS measurement

 2 , 7 - Dichlorodihydrofluorescein diacetate (DCFH/DA) • DCFH/DA diffuses through the cell

2 , 7 - Dichlorodihydrofluorescein diacetate (DCFH/DA) • DCFH/DA diffuses through the cell membrane where it is enzymatically deacetylated by intracellular esterases to the more hydrophilic nonfluorescent reduced dye dichlorofluorescein. • In the presence of reactive oxygen metabolites, DCFH is rapidly oxidized to DCF. • DCF, excitated with 503 nm and emission at 523 nm.

DCFH/DA • Hydroxyl radical, hydrogen peroxide and perhaps a ferryl species, but not superoxide,

DCFH/DA • Hydroxyl radical, hydrogen peroxide and perhaps a ferryl species, but not superoxide, may oxidize DCFH. • The intracellular fluorescent measurements using dichlorofluorescein diacetate may reflect the ability of the test agent or toxicant to generate hydroxyl radical.

DCFH/DA • • • MW 487. 3 Dissolved in 50% methanol Did not dissolved

DCFH/DA • • • MW 487. 3 Dissolved in 50% methanol Did not dissolved in H 2 O or DMSO

Measurement of intracellular H 2 O 2 • Cells incubated with 5 m. M

Measurement of intracellular H 2 O 2 • Cells incubated with 5 m. M dihydrorhodamine 123 for 45 min • PBS wash • Reduced form dihydrorhodamine 123 is oxidized by intracellulr H 2 O 2 to rhodamine 123 • Rhodamine 123 – 485 nm excitation – 530 nm emission

Hydroethidium • Measure superoxide anion concentration • Superoxide anion can be measured by hydroethidium

Hydroethidium • Measure superoxide anion concentration • Superoxide anion can be measured by hydroethidium oxidation into ethidium

Dihydroethidium • Detect superoxide anion Dihydroethidium Oxidation Ethidium Blue fluorescent Red fluorescent Absorption/Emission 355/420

Dihydroethidium • Detect superoxide anion Dihydroethidium Oxidation Ethidium Blue fluorescent Red fluorescent Absorption/Emission 355/420 nm 518/605 nm

Oxidative stress and aging

Oxidative stress and aging

Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the

Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals • Oxidative damage marker 8 -oxo-7, 8 -dihydro-2’deoxyguanosine (8 -oxod. G) in mitochondrial DNA is inversely correlated with maximum life span in the heart and brain of mammals. This inverse relationship is restricted to mt. DNA, not in nuclear DNA.

Doxorubicin-induced Apoptosis is associated with increased transcription of endothelial nitric-oxide synthase • Redox activation

Doxorubicin-induced Apoptosis is associated with increased transcription of endothelial nitric-oxide synthase • Redox activation of DOX by e. NOS – The reductase domain of endothelial nitricoxide synthase (e. NOS) activates doxorubicin (DOX) by a reductive activation and forming semiquinone and superoxide • DOX-induced apoptosis is linked to the redox activation of DOX by e. NOS

 • DOX-induced increase e. NOS transcription and protein expression in bovine aortic endothelial

• DOX-induced increase e. NOS transcription and protein expression in bovine aortic endothelial cells (BAEC). • DOX-induced H 2 O 2 formation is responsible for the increased transcription of e. NOS. Treatment with antioxidants restored the levels of antiapoptotic proteins (Hsp 70 and Bcl-2) in DOXtreated BAEC. • DOX-induced intracellular oxidative stress was inhibited by antisense e. NOS oligonucleotide and antioxidant treatment.

NF B and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells

NF B and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells • Oxidative challenges lead to an increase in antioxidant enzymes, particularly glutathione peroxidase (Gpx) and catalase (CAT) in mouse skeletal muscle • Mouse Gpx and CAT genes revealed putative binding motifs for NF B and AP-1

NF B and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells

NF B and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells • Oxidative stress led to increases in the DNA binding of NF B in differentiated muscle cells. The NF B complexes included a p 50/p 65 heterodimer, a p 50 homodimer, and a p 50/Rel. B heterodimer • Ap-1 is activated, but with slower kinetics than that of NF B

Does oxidative damage to DNA increase with age? • The levels of 8 -oxo-2

Does oxidative damage to DNA increase with age? • The levels of 8 -oxo-2 -deoxyguanosine (oxo 8 d. G) in DNA isolated from tissues of rodents (male F 344 rats, male B 6 D 2 F 1 mice, male C 57 BL/6 mice, and female C 57 BL/6 mice) of various ages were measured. • Oxo 8 d. G was measured in nuclear DNA (n. DNA) isolated from liver, heart, brain, kidney, skeletal muscle, and spleen and in mitochondrial DNA (mt. DNA) isolated from liver.

 • A significant increase in oxo 8 d. G levels in n. DNA

• A significant increase in oxo 8 d. G levels in n. DNA with age in all tissues and strains of rodents studied. • Age-related increase in oxo 8 d. G in mt. DNA isolated from the livers of the rats and mice.

Assay for protein oxidation • 2, 4 -dinitrophenyl hydrazine (DNPH) • carbonyl group in

Assay for protein oxidation • 2, 4 -dinitrophenyl hydrazine (DNPH) • carbonyl group in oxidized protein (10 g ) + DNPH Hydrazone derivatives • SDS-PAGE • Transfer to NC paper • React with anti-dinitrophenylhydrazine antibody • Ref: J. Invest. Dermatol. 112: 1480 -1487 (2004)

Assay for 8 -OHd. G • Cells cytospun to slide • Fixed in methacarn

Assay for 8 -OHd. G • Cells cytospun to slide • Fixed in methacarn (methanol/chloroform/acetic acid, 6/3/1, v/v) for 1 h, RT • Endogenous peroxidase block with H 2 O 2 in methanol 30 min • Nonspecific binding with 10% normal goat serum in Tris-buffered saline 15 mins (150 m. M Tris/HCl and 150 m. M Na. Cl, p. H 7. 6) • Cells treated with proteinase K (20 mg/ml in PBS) 15 min • Cells reacted with anti-8 -OHd. G monoclonal antibody

DNPH reacts with carbonyl group in oxidized protein

DNPH reacts with carbonyl group in oxidized protein

Oxidative stress and diseases

Oxidative stress and diseases

Oxidative damage is the earliest event in Alzheimer disease • A significant increase of

Oxidative damage is the earliest event in Alzheimer disease • A significant increase of an oxidized nucleoside derived from RNA, 8 hydroxyguanosine (8 OHG), and an oxidized amino acid, nitrotyrosine in vulnerable neurons of patients with Alzheimer disease (AD). • The increased oxidative damage is an early event in AD that decreases with disease progression and lesion formation.

Reactive oxygen species increase risk of disease through damage to key biological structures

Reactive oxygen species increase risk of disease through damage to key biological structures

Free radicals in disease • The formation of ROS is a feature of many

Free radicals in disease • The formation of ROS is a feature of many degenerative diseases, such as atherosclerosis and neurodegeneration

ROS involved in stroke • Stroke is a severe and prevalent syndrome for which

ROS involved in stroke • Stroke is a severe and prevalent syndrome for which there is a great need for treatment, including agents to block the cascade of brain injury that occurs in the hours after the onset of ischemia. ROS have been implicated in this destructive process • EUK-134, a newly reported salenmanganese complex having greater catalase and cytoprotective activities and equivalent SOD activity compared with the prototype EUK-8

Small molecules mimicing antioxidant enzymes • Mn(II) complex M 40403 (a synzyme) – possesses

Small molecules mimicing antioxidant enzymes • Mn(II) complex M 40403 (a synzyme) – possesses SOD activity approaching that of the native Mn-SOD enzyme – possessing outstanding chemical and biological stability • Removes superoxide without interfering with other relevant biological oxidants, such as nitric oxide, peroxynitrite, or hydrogen peroxide