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) • O2-. (superoxide radical) • H2O2 (hydrogen peroxide) • NO. (nitric oxide) • Oxidative stress • Oxidative 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. • 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 semiquinone radical • Microsomal (ER) electron transport system • Superoxide radical and H2O2 • Arachidonic acid metabolism • Reactions within peroxisome • Superoxide radical and H2O2
H2O2 and O2-. may diffuse from their subcellular sites of production and affect the whole cell • H2O2 can cross biological membranes
NO. • Small • Gas • Synthesize by nitric oxide synthase (NOS) • nNOS: constitutive expression • eNOS: constitutive expression • iNOS: inducible
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 • Protein-thiol • Cerutoplasmin: important in reducing Fe3+ release from ferritin • Antioxidative enzyme
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 • Hydroxyl radicals • Lipid hydroperoxides
ROS act as second messengers • Ligand-receptor interactions produce ROS and that antioxidants block receptor-mediated 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 ROS Tumor necrosis factor- H2O2/HO Interleukin 1 H2O2/O2- Transforming growth Factor-1 H2O2 Platelet derived growth factor H2O2 Insulin H2O2 Angiotension II H2O2/O2- Vitamin D3 O2- Parathyroid hormone O2-
TNF activates oxidative stress-responsive transcription factors, NF-B and AP-1, and also induces apoptosis.
ROS induce apoptosis • Both H2O2 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 N-acetylcysteine, catalase, vitamin E, and 2-mercaptoethanol
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. • H2O2 activates the DNA binding activity of p53. P53 is required for the induction of apoptosis.
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, 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 in H2O or DMSO
Measurement of intracellular H2O2 • Cells incubated with 5 mM dihydrorhodamine 123 for 45 min • PBS wash • Reduced form dihydrorhodamine 123 is oxidized by intracellulr H2O2 to rhodamine 123 • Rhodamine 123 • 485 nm excitation • 530 nm emission
Hydroethidium • Measure superoxide anion concentration • Superoxide anion can be measured by hydroethidium oxidation into ethidium
Dihydroethidium • Detect superoxide anion Oxidation Dihydroethidium Ethidium Blue fluorescent Absorption/Emission 355/420 nm Red fluorescent Absorption/Emission 518/605 nm
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-oxodG) in mitochondrial DNA is inversely correlated with maximum life span in the heart and brain of mammals. This inverse relationship is restricted to mtDNA, not in nuclear DNA.
Doxorubicin-induced Apoptosis is associated with increased transcription of endothelial nitric-oxide synthase • Redox activation of DOX by eNOS • The reductase domain of endothelial nitric-oxide synthase (eNOS) activates doxorubicin (DOX) by a reductive activation and forming semiquinone and superoxide • DOX-induced apoptosis is linked to the redox activation of DOX by eNOS
DOX-induced increase eNOS transcription and protein expression in bovine aortic endothelial cells (BAEC). • DOX-induced H2O2 formation is responsible for the increased transcription of eNOS. Treatment with antioxidants restored the levels of antiapoptotic proteins (Hsp70 and Bcl-2) in DOX-treated BAEC. • DOX-induced intracellular oxidative stress was inhibited by antisense eNOS oligonucleotide and antioxidant treatment.
NFB 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 NFB and AP-1
NFB and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells • Oxidative stress led to increases in the DNA binding of NFB in differentiated muscle cells. The NFB complexes included a p50/p65 heterodimer, a p50 homodimer, and a p50/RelB heterodimer • Ap-1 is activated, but with slower kinetics than that of NFB
Does oxidative damage to DNA increase with age? • The levels of 8-oxo-2-deoxyguanosine (oxo8dG) in DNA isolated from tissues of rodents (male F344 rats, male B6D2F1 mice, male C57BL/6 mice, and female C57BL/6 mice) of various ages were measured. • Oxo8dG was measured in nuclear DNA (nDNA) isolated from liver, heart, brain, kidney, skeletal muscle, and spleen and in mitochondrial DNA (mtDNA) isolated from liver.
A significant increase in oxo8dG levels in nDNA with age in all tissues and strains of rodents studied. • Age-related increase in oxo8dG in mtDNA isolated from the livers of the rats and mice.
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-OHdG • Cells cytospun to slide • Fixed in methacarn (methanol/chloroform/acetic acid, 6/3/1, v/v) for 1 h, RT • Endogenous peroxidase block with H2O2 in methanol 30 min • Nonspecific binding with 10% normal goat serum in Tris-buffered saline 15 mins (150 mM Tris/HCl and 150 mM NaCl, pH7.6) • Cells treated with proteinase K (20 mg/ml in PBS) 15 min • Cells reacted with anti-8-OHdG monoclonal antibody
Oxidative damage is the earliest event in Alzheimer disease • A significant increase of an oxidized nucleoside derived from RNA, 8-hydroxyguanosine (8OHG), 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 throughdamage to key biological structures
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 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 salen-manganese complex having greater catalase and cytoprotective activities and equivalent SOD activity compared with the prototype EUK-8
Small molecules mimicing antioxidantenzymes • Mn(II) complex M40403 (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