Oxidative Stress

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老化與抗氧化能力及其相關分子 檢測

曾婉芳 教授

Oxidative Stress

• Reactive oxygen species (ROS)

• ROS and oxidative stress

• Antioxidant system

• Oxidative damage

• Oxidative stress and aging

• Markers of oxidative damage

• Markers of aging

Reactive oxygen species (ROS)

• ROS

– OH^. (hyroxyl radical)

– O₂^-. (superoxide radical) – H₂O₂ (hydrogen peroxide) – NO^. (nitric oxide)

• Oxidative stress

• Oxidative damage

ROS generated in cells and tissues

Reactive nitrogen species (RNS)

generated in cells and tissues

Toxic effects of ROS

• Protein oxidation

• Lipid peroxidation

• Nucleic acids damage

– Double-strand DNA breaks – Single-strand DNA breaks – DNA bases change

• 8-oxoguanine

• 8-hydroxyguanosine

• Thymine glycol

Consequences of ROS and RNS on protein function and fate

• Protein oxidation

– Permanent loss of protein function

– Degradation of the damaged proteins by proteasome and other proteases

– Accumulation of damaged proteins

Protein oxidation in oxidative stress

• Attack of ROS on amino acid

– Generating oxo-, sulfo-, hydroxy-, chloro-, and nitro-derivatives

• Oxidative attack of polypeptide backbone is initiated by the ^•OH-dependent abstraction of the α-hydrogen atom of an amino acid residue to form a carbon-centered radical.

Proteins vulnerable to oxidative damage

• Not all proteins are uniformly susceptible to oxidative damage.

• Mitochondrial aconitase, adenine nucleotide translocase, glutamine synthetase and creatine kinase are particularly vulnerable to oxidative damage.

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 ROS

• Mitochondria

– Complex I and III of electron transport chain

• Endoplasmic reticulum – Cytochrome P450

• Plasma membrane – NADPH oxidase

• Cytosol

– Xanthine oxidase

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

Superoxide production in mitochondria

• At complex I (NADH coenzyme Q reductase) – Iron–sulphur centres or the ‘active site

flavin’

• At complex III (bc1 complex)

– Cytochrome b rather than ubisemiquinone

Intracellular sources of free radicals

• In cytosol

– Xanthine oxidase oxidizes xanthine and generates H₂O₂

– Amino acid oxidase generates H₂O₂ as their ordinary products

• H₂O₂ and O₂^-. may diffuse from their

subcellular sites of production and affect the whole cell.

• H₂O₂ can cross biological membranes

NO

synthesis

Antioxidative system

• Antioxidant

– Glutathione, GSH – Vitamin C, E

– Cysteine

– Protein-thiol

– Cerutoplasmin: important in reducing Fe³⁺ release from ferritin

• Antioxidative enzyme

Glutathione (GSH)

Antioxidative enzyme

• Catalase

• Superoxide dismutase

• Glutathione peroxidase

• Glutathione reductase

• Gluththione S-transferase

• Glucose-6-phosphate dehydrogenase

• DT-diaphorase

Catalase (EC 1.11.1.6)

• 2H₂O₂ → 2H₂O＋O₂ catalase

• A homotetrameric haeminenzyme, 240 kD

• One of the most efficient enzymes known

• It is so efficient that it cannot be saturate by H₂O₂ at any concentration

Superoxide dismutase (SOD. EC 1.15.1.1)

• Human SOD

– Cytosolic CuZn-SOD

– Mitochondrial SOD: MnSOD – Extracellular SOD

• 2O₂^-. ＋ 2H^＋ → H₂O₂ ＋ O₂ SOD

Extracellular superoxide dismutase (EC-SOD)

• A secretory, tetramer

• Copper and zinc containing glycoprotein

• In extracellular fluids

• The majority of the SOD activity of plasma, lymph, and synovial fluid

• Regulated by cytokines

Glutathione peroxidase (GP, EC 1.11.1.19)

glutathione peroxidase

ROOH → ROH＋H₂O

2GSH GSSG

Glutathione peroxidase (GP)

• GP contains covalently bound Se (selenium) in the form of selenocysteine

GPX isoenzymes

• Cytosolic GPX (cGPX)

• Mitochondrial GPX (GPX1) – found in most tissues

– Predominantly present in erythrocytes, kidney, and liver

• Phospholipid hydroperoxide glutathione peroxidase GPX4 (PHGPX)

• Cytosolic GPX2 (GPX-G1)

• Extracellular GPX3 (or GPX-P)

• GPX5

– Expressed specifically in mouse epididymis, selenium-independent

Glutathione reductase (GR)

glutathione reductase

GSSG＋H^＋

→

NADPH NADP^＋

Glucose-6-phosphate dehydrogenase (G6PD)

glucose-6-phosphate dehydrogenase, Mg²⁺

Glucose-6-phosphate → 6-phosphoglucono-δ-lactone NADP^＋ NADPH

DT-diaphorase

• NAD(P)H：(quinone acceptor) oxidoreductase (EC 1.6. 99.2)

• In cytosol

• Two electron transfer of quinone compounds Quinone → Hydroquinone

Glutathione S-transferase (GST)

• Detoxification of toxic compounds (RX) to increase the solubility of the compound

• The less toxic derivative of the original

compound can then be excreted in the urine.

Detoxification by glutathione S-transferase

(GST)

Heme oxygenase

• Heme → biliverdin →bilirubin

• A major stress protein induced in cells response to oxidant stress

• Bilirubin is an efficient plasma or serum scavenger of singlet ¹O₂, O₂^-., and peroxy radicals.

Oxidants as stimulators of signal transduction

• Oxidants

– Superoxide

– Hydrogen peroxide – Hydroxyl radicals

– Lipid hydroperoxides

ROS as second messengers

• Generation of ROS by cytokines

Ligand ROS

Tumor necrosis factor-α H₂O₂/HO^⋅⋅⋅⋅

Interleukin 1β H₂O₂/O₂^{-⋅⋅⋅⋅}

Transforming growth Factor-β1 H₂O₂ Platelet derived growth factor H₂O₂

Insulin H₂O₂

Angiotension II H₂O₂/O₂^{-⋅⋅⋅⋅}

Vitamin D₃ O₂^{-⋅⋅⋅⋅}

Parathyroid hormone O₂^{-⋅⋅⋅⋅}

ROS detection

• Chemiluminescence of luminol and lucigenin

• Cytochrome c reduction

• 2’-7’-Dichlorodihydrofluorescence diacetate (DCFH-DA)

Chemiluminescence of luminol and lucigenin

• Cell permeable method for ROS detection

• Luminol is sensitive to H₂O₂ and peroxynitrite, but not sensitive to superoxide.

• Lucigenin is specific for superoxide.

Luminol-dependent chemiluminescence assay

• Based on the oxidation of luminol by sodium hypochlorite (NaOCl). H₂O₂ reacts with this oxidized product, generating an excited

molecule capable of luminescence

• Specific for H₂O₂

• Detect nM H₂O₂

DCFH-DA

• DCFH-DA, a cell permeable, nonfluorescent precursor of DCF

• Intracellular esterases cleave DCFH-DA at the two ester bonds, produce a relatively polar and cell-membrane impermeable product, H₂DCF.

• H₂DCF, can be oxidized by H₂O₂, yields the fluorescent DCF.

DCFH-DA

2′,7′- Dichlorodihydrofluorescein diacetate (DCFH/DA)

• In the presence of reactive oxygen metabolites, DCFH is rapidly oxidized to DCF.

• DCF

– excitation with 503 nm – emission at 523 nm

• Hydroxyl radical, hydrogen peroxide and

perhaps a ferryl species, but not superoxide, may oxidize DCFH.

Dihydroethidium

• Detect superoxide anion

Dihydroethidium Oxidation Ethidium

Blue fluorescent

Absorption/Emission 355/420 nm

Red fluorescent

Absorption/Emission 518/605 nm

superoxide anion

Aging and oxidative stress

• Mammalian aging is associated with

accumulation of oxidative damage in DNA, proteins, and lipids.

Mitochondrial DNA mutation

• Mitochondrial DNA (mtDNA) is more sensitive to oxidative stress.

• mtDNA, unlike nuclear DNA, is not protected by histone proteins.

MDA: malondialdehyde DBI: double bond index

Oxidative DNA damage measurements in non-cancerous pathological conditions

• Parkinson’s disease (PD)

– DNA levels of 8-OH-dG significantly elevated (P

= 0.0002) in substantia nigra of PD brains

• Alzheimer’s disease

– Higher levels of 8-OH-dG in cortex and cerebellum of AD patients vs.controls

• Systemic lupus erythematosus (SLE)

– PBMC levels of 8-OH-dG significantly higher in SLE patients vs.controls (P = 0.0001)

Dual role of mitochondrial ROS production as a signaling mechanism and as a cause of

age-associated cellular damage

Aging marker

Senescence-associatedβ-galactosidase (SA-β-gal) staining

• Lysosomalβ-galactosidase of aging cells released to cytosol

• Cytosolic β-galactosidase increased during aging

Senescence-associatedβ-galactosidase (SA-β-gal) staining

• β-Galactosidase is an enzyme that catalyzes the hydrolysis of β-galactosides, including lactose.

• β-galactosidase cleaved β-galactosidic bond of X-gal (5-bromo-4-chloro-3-indolyl-β-D- galactoside) , and 5-bromo-4-chloro-3-indoly

was released.

* Positive reaction: Blue

X-gal

M.W. : 408.6

Formula:C₁₄ H₁₅ Br Cl N O₆ β

ββ

β- galactosidic bond

Senescence-associated β-galactosidase (SA-β-gal) staining

Young HCA2 cell Senescent HCA2 cell

Oxidative stress and SA β -gal

• Treatment of chondrocytes for 4 days with 20 μM tert-butylhydroperoxide (4 h per day)

caused increased expression of senescence- associated β-galactosidase and DNA

oxidation, and decreased mitochondrial function.

Chronic oxidative stress compromises

telomere integrity and accelerates the onset

of senescence in human endothelial cells

• Human umbilical vein endothelial cells were treated to 0.1 µM tert-butyl hydroperoxide

(substrate of glutathione peroxidase), or 10 µM L- buthionine-[S,R]-sulphoximine (inhibitor of

glutathione synthesis).

• Both treatments induced intracellular oxidative stress but had no cytotoxic or cytostatic effects.

• Treated cells entered senescence prematurely (30 versus 46 population doublings), as determined by senescence-associated β-galactosidase staining.

Glucose-6-phosphate dehydrogenase- deficient cells show an increased

propensity for oxidant-induced senescence

• G6PD-deficient fibroblasts undergo premature cellular senescence.

• A significant increase in the level of 8- hydroxy-2-deoxyguanosine (8-OHdG).

• G6PD-deficient cells had an increased

propensity for H₂O₂-induced senescence with senescent phenotypes as large, flattened

morphology and increased senescence- associated β-galactosidase.

Ki 67

• A proliferation marker

• Expressed in G1, S, G2, M phase

• Do not express in G0

• Control of the higher order chromatin structure

• Detection by Anti-Ki-67 Ab

Melatonin and human aging

• Available in some countries (e.g. USA, Argentina, and Poland) as a food supplement, and is often

advertised as a ‘rejuvenating’ agent.

• Changes in melatonin secretion during life-span

• Significance of melatonin secretion decline for reduced antioxidant protection in elderly.

• Melatonin exerts immunoenhancing action, both in animals and in humans.

Circadian profiles of serum melatonin concentrations at various age

gray area—darkness

Melatonin

• A potent free radical scavenger and antioxidant that scavenges especially highly toxic

hydroxyl radicals.

• Stimulates a number of antioxidative enzymes

• Melatonin is both lipophylic and hydrophilic

and diffuses widely into cellular compartments, thus providing on-site protection against free

radical mediated damage to biomolecules.

Melatonin

• The only antioxidant known to decrease substantially after middle age, and this

decrease closely correlates with a decrease in total antioxidant capacity of human serum with age.

Significance of melatonin in age-related diseases

• Neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease

– high vulnerability of the central nervous system to oxidative attack

Markers of oxidative damage

• Protein oxidation – protein carbonyls

• Lipid peroxidation

– Malondialdehyde (MDA), 4- hydroxynonenal (HNE) and 4- hydroxyhexenal (HHE)

• DNA damage – 8-OHdG

Protein oxidation in aging

• The most widely studied marker of protein oxidation is protein carbonyl groups.

• Direct oxidation of protein side chains

– Oxidation of the side chains of lysine, proline, arginine, and threonine residues

• Addition carbonyl groups into proteins

– By addition reactions of 4-hydroxynonenal, a product of lipid peroxidation

Measurement of protein carbonyls

• Reaction of protein carbonyls with 2,4-

dinitrophenylhydrazine (DNPH) to form the 2,4-dinitrophenylhydrazone

• The levels of the protein carbonyl levels are measured by the absorbance of the 2,4-

dinitrophenylhydrazone at 370 nm.

Urinary 8-OHdG

• A marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics

• Detection by HPLC or ELISA

Lipid peroxidation products

• Malondialdehyde (MDA), 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE)

• HNE

Lipid peroxidation measured by thiobarbituric acid assay

• Thiobarbituric acid assay

– reaction of aldhydic groups on products (e.g., malondialdehyde (MDA) and 4-hydroxy-2- nonenol (4-HNE))

– aldhydic groups on products arose from free radical-initiated oxidative damage of

polyunsaturated fatty acids

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