老化與抗氧化能力 及其相關分子檢測

老化與抗氧化能力及其相關分子檢測

Dr.曾婉芳

Oxidative stress

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

Intracellular sources of free radicals

• In cytosol

– Xanthine oxidase oxidizes xanthine and generates H2O2

– Amino acid oxidases generates H2O2 as their ordinary products

• H2O2 and O2-. may diffuse from their subcel

lular sites of production and affect the whole cell

• H2O2 can cross biological membranes

NO. synthesis

Reactive nitrogen species (RSN)

• 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

Dietary oxidants

• Generation of ROS

• ROS are reduced or inactivated through the generation of a disulfur bond between two glutathione molecules to form oxidized glutathione

Xenobiotics

• Man-made compounds with chemical structures foreign to a given organism

• Induce cancer

• Glutathione is involved in the conjugation of epoxides to less toxic compounds that will be eventually excreted

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• Subunit 60 kD• Four ferriprotoporphyrin groups• 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 ＋ O

2

superoxide dismutase

Manganese SOD (MnSOD)

• A homotetramer (96 kDa) containing one manganese atom per subunit

• Cycles from Mn(III)–Mn(II) and back to Mn(III) during the dismutation of superoxide

Cytosolic CuZn-SOD

• Two identical subunits of about 32 kDa

• Each containing a metal cluster, the active site, constituted by a copper and a zinc atom bridged by a common ligand: His 61

• Inactivation of copper- and zinc-containing SOD by H2O2 is the consequence of several sequential reactions

Inactivation of cytosolic CuZn-SOD by H2O2

• Reduction of the active site Cu(II) to Cu(I) by H2O2

• Oxidation of the Cu(I) by a second H2O2, thus generating a powerful oxidant, which may be Cu(I)O, Cu(II)OH or Cu(III)

• Oxidation of the histidine, causing loss of SOD activity

Extracellular superoxide dismutase (EC-SOD)

• A secretory, tetrameric, copper and zinc containig glycoprotein

• High affinity for certain glycosaminogycans such as heparin and heparan sulfate

• In the intersticial spaces of tissues• In extracellular fluids, accounting for the m

ajority of the SOD activity of plasma, lymph, and synovial fluid

EC-SOD

• Not induced by its substrate or other oxidants (xanthine oxidase plus hypoxanthine, paraquat, pyrogallol, a-naphthoflavone, hydroquinone, catechol, Fe2+, Cu2+, buthionine sulphoximine, diethylmaleate, t-butyl hydroperoxide, cumene hydroperoxide, selenite, citiolone and high oxygen partial pressure)

• Its regulation in mammalian tissues primarily occurs in a manner coordinated by cytokines, rather than as a response of individual cells to oxidants

Nickel superoxide dismutase(Ni-SOD)

• Purified from the cytosolic fraction of Streptomyces sp. and Streptomyces coelicolor

• Four identical subunits of 13.4 kDa, stable at pH 4.0–8.0, and up to 70°C

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, kidne

y, 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

GPX

• cGPX and GPX1 reduce fatty acid hydroperoxides and H2O2 at the expense of GSH

• Cytosolic GPX2 (GPX-G1) and extracellular GPX3 (GPX-P) are poorly detected in most tissues except for the gastrointestinal tract and kidney, respectively.

GPX1

• 80 kD, contains one selenocysteine (Sec) residue in each of the four identical subunits, which is essential for enzyme activity

• The principal antioxidant enzyme for the detoxification of H2O2 has for a long time been considered to be GPX, as catalase has much lower affinity for H2O2 than GPX

PHGPX

• Found in most tissues• Highly expressed in renal epithelial cells an

d testes• Located in both the cytosol and the membr

ane fraction• Directly reduce the phospholipid hydropero

xides, fatty acid hydroperoxides, and cholesterol hydroperoxides that are produced in peroxidized membranes and oxidized lipoproteins

Tissue-specific functions of individual glutathione peroxidases

• All glutathione peroxidases reduce hydrogen peroxide and alkyl hydroperoxides at expense of GSH

• Four glutathione peroxidases isozymes

1. Classical glutathione peroxidase (cGPx)

2. Gastrointestinal glutathione peroxidases

(GI-GPx)

3. Plasma GPx (pGPx)

4. Phospholipid hydroperoxide glutathione peroxidases (PHGPx)

Classical glutathione peroxidase (cGPx)

• Ubiquitously distributed

• Reduces only soluble hydroperoxides, such as H2O2, and some organic hydroperoxid

es, such as hydroperoxyl fatty acids, cumene hydroperoxide, or t-butyl hydroperoxide

Gastrointestinal glutathione peroxidases

(GI-GPx)• Expressed in gastrointestinal tract

• Provides a barrier against hydroperoxides derived from the diet or from metabolism

of ingested xenobiotics

• Substrate specificity is similar to that of cGPx

 Plasma GPx (pGPx)

• Expressed in tissues in contact with body fluids, e.g., kidney, ciliary body, and maternal/fetal interfaces

Phospholipid hydroperoxide glutathione peroxidases (PHGPx)

• Protects membrane lipids• Reduces hydroperoxides of more complex lipi

ds like phosphatidylcholine hydroperoxide• Reduces hydroperoxo groups of thymine, lipop

roteins, and cholesterol esters• Unique in acting on hydroperoxides integrated

in membranes• Silence lipoxygenases• Becomes an inactive structural component of t

he mitochondrial capsule during sperm maturation

Glutathione reductase (GR)

glutathione reductase

GSSG ＋ H ＋ 2GSH

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 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 cytokinesLigand 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-

Oxidative stress and mitochondria

• During the course of normal oxidative phosphorylation, between 0.4 and 4% of all oxygen consumed is converted into the superoxide free radical (O2

-.).

Intracellular sources of ROS

• Mitochondria– Complex I and III of electron transport chain

• Endoplasmic reticulum– Cytochrome P450

• Plasma membrane– NADPH oxidase

• Cytosol– Xanthine oxidase

ROS detection

• Chemiluminescence of luminol and lucigenin

• Cytochrome c reduction

• Ferrous oxidation of xylenol orange

• 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 CL assay

• The assay is 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 imperable product, H2DCF

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

DCFH-DA

  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

Hydroethidium

• Measure superoxide anion concentration

• Superoxide anion can be measured by hydroethidium oxidation into ethidium

Dihydroethidium

• Detect superoxide anion

Dihydroethidium EthidiumOxidation

Blue fluorescent

Absorption/Emission

355/420 nm

Red fluorescent

Absorption/Emission

518/605 nm

O2.- production in electron transport chain

• Superoxide anions can be produced at both complex I and III

• Semiquinone formation at both complex I and III results in the production of superoxide anions

Mitochondria – the major sites of cellular ROS production

• Approximately 0.2–2% of the oxygen taken up by cells is converted by mitochondria to ROS, mainly through the production of superoxide anion

• The two major sites of superoxide production are at complex I and complex III

Sites of superoxide formation in the respiratory chain

Superoxide production in mitochondria

• At complex I (NADH coenzyme Q reductase)

– Iron–sulphur centres or the ‘active site flavin’

• At complex III (bc1 complex)

– was cytochrome b rather than ubisemiquinone

Aging and oxidative stress in mammals and birds

• Both long-lived and calorie-restricted animals constitutively have low levels of production of mitochondrial reactive oxygen species (ROS), which could be responsible for their low rate of accumulation of mitochondrial DNA (mtDNA) mutations, and thus for their low rate of aging.

Aging and oxidative stress in mammals and birds

• Long-lived species also have low degrees of fatty acid unsaturation (DBI, double bond index) in their cellular membranes, and thus lower levels of lipid peroxidation (MDA, malondialdehyde) and lipoxidation-derived protein modification (Prot. ox.). This lower lipid peroxidation can also be partially responsible for the lower levels of oxidative damage in their mtDNA.

Mitochondrial DNA

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

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

DNA base damage

DNA base damage

Product formation from the C5-OH-adduct radical of cytosine in the absence of oxygen

Product formation from the C5- and C6-OH-adduct radicals and allyl radical of thymine

Product formation from the C5- and C6-OH-adduct radicals and allyl radical of thymine

Product formation from the C5- and C6-OH-adduct radicals of cytosine in the

presence of oxygen

Product formation from the C5- and C6-OH-adduct radicals of cytosine in the

presence of oxygen

Reactions of •OH with purines

Reactions of C4- and C5-OH-adduct radicals of guanine

Product formation from the C8-OH-adduct radical of guanine in the absence of oxygen

Major products of oxidative damage to the DNA bases-1

Major products of oxidative damage to the DNA bases-2

Major products of oxidative damage to the DNA bases-3

Major products of oxidative damage to the DNA bases-4

Major products of oxidative damage to the DNA bases-5

Oxidative DNA damage measurements in cancerous/pre-cancerous conditions

• Acute lymphoblastic leukaemia (ALL)

– Lymphocyte DNA levels of FapyGua, 8-OH-Gua, FapyAde, 8-OH-Ade, 5-OH-Cyt, 5-OH-5-MeHyd and 5-OH-Hyd significantly (P < 0.05) elevated in ALL compared to control subjects.

Breast cancer

• Significantly higher (P < 0.0001) levels of 8-OH-dG in DNA from tumour, compared non-tumour tissue

Cervical cancer

• Levels of 8-OH-dG significantly increased (P < 0.001) in DNA from low-grade and high-grade levels of dysplasia, compared to normal, although this did not correlate with human papillomavirus status.

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

Oxidative DNA damage measurements in non-cancerous pathological conditions

• Systemic lupus erythematosus (SLE)

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

– Titres of serum autoantibodies to 5-OHMeUra significantly elevated in SLE

Oxidative DNA damage measurements in non-cancerous pathological conditions

• Rheumatoid arthritis (RA)

– Levels of urinary 8-OH-dG significantly elevated in RA patients (P < 0.001), compared to control subjects

– PBMC levels of 8-OH-dG significantly higher in RA patients vs. controls

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

age-associated cellular damage

Aging marker

Senescence-associated -galactosidase(SA -gal)

Ki 67

• Expressed in G1, S, G2, M phase

• Do not express in G0

PCNA

P105

• Expressed in G1, S, G2, M phase

– G1 and S phase: in Nucleus

– G2 and M phase: in cytoplasm

• Do not express in G0

Redox control of cellular scenescence

• Mammalian aging is associated with accumulation of oxidative damage in DNA, proteins, and lipids.

Telomere shortening

• Telomeres, the repetitive DNA and specialized proteins that cap the ends of the linear chromosome, prevent chromosome fusion and genomic instability.

• Telomerase, the enzyme that synthesizes telomeric DNA de novo, is absent from most normal somatic cells.

• Telomeres shorten with cell division.

Senescence is due to downregulationof positive-acting cell cycle regulatory genes

• c-fos proto-oncogene

• Genes for Cdc2 and cyclin A and E, components of CDKs, genes for Id1 and Id2 inhibitors of HLH-transcription factors

• E2F1 transcription factor

Upregulation of cell growth inhibitors

• Elevated levels of growth inhibitors p21, p16, and in some cases, p27

ROS generated in cells and tissues

Reactive nitrogen species (RNS)generated in cells and tissues

Consequences of ROS/RNS and oxidative/nitrosative stress on protein function and fat

e

• Irreversible modifications are usually associated with permanent loss of protein function and may lead to the degradation of the damaged proteins by proteasome and other proteases or to their progressive accumulation.

Oxidative/nitrosative modifications of protein Cys residues

• ROS/RNS may induce the formation of mixed disulphides between protein thiol groups (PSH) and GSH to form S-glutathionylated proteins (PSSG).– PSH may be initially “activated” by oxidative/nitrosative m

odifications to give thyil radical (PS·), sulphenic acid (PSOH), or protein S-nitrosothiol/S-nitrosated protein (PSNO). These modifications may be either stabilized as such or react with GSH to the mixed disulphide (PSSG). All these modifications are reversible and can be reduced back by increases in the GSH/GSSG ratio, reduced thiols, or enzymatic reactions. Otherwise,

• PSSG may be generated by thiol/disulphide exchange reaction with GSSG or by reaction with other “reactive” intermediates of GSH, such as GSNO.

Oxidative/nitrosative modifications of protein Cys residues

• PSOH may also be irreversibly oxidised by ROS/RNS to form sulphinic (PSO2H)

• and sulphonic (PSO3H) derivatives, leading to irreversible loss of biological activity. PSH may also be oxidised to

• disulphide both within and between proteins (PSSP). PSSP can be reversed by enzymes (protein disulphide isomerase and thioredoxin/thioredoxin reductase) or reducing agents.

Methionine sulfoxide reductases

• Moskovitz, J.

Biochimica et Biophysica Acta 1703: 213– 219 (2005)

• Enzymes involved in antioxidant defense, protein regulation, and prevention of aging-associated diseases

• Met oxidation may play an important role in the development and progression of neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases.

Methionine and cysteine

• Two sulfur amino acids that are readily oxidized under conditions of oxidative stress.

• Cysteine can be regenerated by a number of non-enzymatic (e.g. glutathione) and enzymatic pathways (e.g. involving NADPH-dependent enzymatic reactions)

• For MetO reduction an addition of Msr enzymes is needed.

Methionine oxidation

• ROS can oxidize Met to methionine sulfoxide (MetO) forming two enantiomers: S-MetO and R-MetO.

• Enzymatic system for reduction of MetO

Methionine sulfoxide reductases (Msr)

Thioredoxin (Trx)

Thioredoxin reductase (Trr)

NADPH• Reduction of free and protein-bound MetO

Methionine sulfoxide reductases

• MsrA protein reduces S-MetO

• MsrB protein reduces R-MetO

MsrA and aging

• Abolish the MsrA gene in mice shortened their life span both under normoxia and hyperoxia (100% oxygen)

Proteins function regulated by methionine oxidation and reduction

• Potassium channel of the brain

• Calmodulin

• Reversal methionine oxidation may play an important role in regulation of protein’s function either directly or mediated by signal transduction pathways.

Melatonin, human aging, and age-related diseases

• Experimental Gerontology 39: 1723–1729 (2004)

Melatonin

• Available in some countries (e.g. USA, Argentina, and Poland) as a food supplement or an over the counter drug, and is often advertised as a ‘rejuvenating’ agent.

Changes in melatonin secretion during life-span

• In mammals, melatonin concentrations exhibit a clear circadian rhythm, with low values during the daytime and high values (10-15X increase) at night.

• Circadian rhythms are present in all living organisms, from unicellular algae to man.

Circadian profiles of serum melatonin concentrations at various age

gray area—darkness

Melatonin

• Pineal gland is to adjust the phase and synchronize internal rhythms by the periodic release of melatonin.

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

Significance of melatonin secretion decline for reduced antioxidant protection in elderly

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

• Oxidative damage plays an important role in the pathogenesis of neurodegenerative diseases characteristic of aged population.

• Neurodegenerative diseases such as Alzheimer’s and Parkinson’s because of high vulnerability of the central nervous system to oxidative attack and neoplastic disease.

Alzheimer’s disease

• Features

– Amyloid- plaques

– Neurofibrillary tangles, and extensive neural loss, particularly in the hippocampus and cerebral cortex

– The neuronal loss is most probably caused by free radicals generated by amyloid- peptide (in particular by its 25–35 amino acid residue)

Alzheimer’s disease and melatonin

• Melatonin may reduce the neurotoxicity of the amyloid- , leading to increased cellular survival.

• Decreased melatonin concentrations were observed in some, but not all, patients suffering from Alzheimer’s disease.

Parkinson’s disease

• Features– Progressive deterioration of dopamine-conta

ining neurons in the pars compacta of the substantia nigra in the brain stem.

• The loss of these neurons is caused by auto-oxidation of dopamine due to relatively high exposure of these neurons to free radicals.

Parkinson’s disease and melatonin

• In experimental animal models of Parkinson’s disease, melatonin administration diminished lipid peroxidation that occurred in the striatum, hippocampus and midbrain after injection of 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine and reduced cytotoxicity of 6-hydroxydopamine

Consequences of ROS/RNS and oxidative/nitrosative stress on protein function and fat

e• ROS/RNS may cause oxidative/nitrosative mo

difications on sensitive target proteins.

• Reversible modifications, usually at Cys and Met residues, may have a dual role of modulation of protein function and protection from irreversible modification.

Oxidatively modified proteins in aging and disease

Protein oxidation

• 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 Michael addition reactions of 4-hydroxynonena

l, a product of lipid peroxidation

Measurement of protein carbonyls

• The most widely utilized measure of protein oxidation

– Reaction of protein carbonyls with 2,4-dinitrophenylhydrazine (DNPH) to form the corresponding hydrazone

– The levels of the protein carbonyl levels are measured by the absorbance of the 2,4-dinitrophenylhydrazone at 370 nm

Measurement of 3-nitrotyrosine

• By using HPLC with the electrochemical detection

• By mass spectroscopy

• By immunohistochemistry

Oxidative damage and aging

• Increases in the intracellular concentrations of oxidized proteins as a function of age.

• Increases in protein carbonyls occur in rat hepatocytes, drosophila, brain, and kidney of mice and in brain tissue of gerbils.

• In humans protein carbonyls increase with age in brain, muscle, and human eye lens.

Oxidative damage and aging

• In drosophila, restricting flying increases life span, and this correlates with reduced protein carbonyls.

• Transgenic mice with a knockout of methionine sulfoxide reductase, which repairs oxidized methione, have a reduced life span and show increased protein carbonyls.

Proteins vulnerable to oxidative damage

• Not all proteins are uniformly susceptible to oxidative damage.

• Mitochondrial aconitase was particularly vulnerable to oxidative damage accompanying aging in drosophila.

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

Alzheimer’s disease

• Neuropathologic hallmarks are senile plaques containing -amyloid and neurofibrillary tangles, which occur in pyramidal neurons of the cerebral cortex and hippocampus.

• Patients taking antioxidant vitamins and anti-inflammatory compounds have a lower incidence of AD.

• Protein carbonyls were significantly increased in both hippocampus and the inferior parietal lobule, but unchanged in the cerebellum, consistent with the regional pattern of histopathology in AD.

Alzheimer’s disease

• Significant decreases in glutamine synthetase and creatine kinase activity.

• Oxidative damage to the glial glutamate transporter

• Increases in protein carbonyls both in neurofibrillary tangles as well as in the cytoplasm of tangle free neurons.

Parkinson’s disease

• The second most common neurodegenerative disease.

• It causes a progressive movement disorder.

• Loss of substantia nigra dopaminergic neurons.

• The histopathologic hallmark is eosinophilic cytoplasmic inclusions in the substantia nigra neurons known as Lewy bodies.

Parkinson’s disease

• Increases in protein carbonyls in all brain regions including the substantia nigra, basal ganglia, globus pallidus, substantia innominata, frontal cortex, and cerebellum.

• Peroxynitrite-induced protein damage

Amyotrophic lateral sclerosis (ALS)

• A rapidly progressive neurodegenerative disease leading to progressive motor weakness and death.

• A loss of motor neurons in both the motor cortex and the spinal cord.

• Increase in protein carbonyls in frontal cortex and in motor cortex

• Increased protein nitration in ALS

Huntington’s disease

• An autosomal dominant inherited neurodegenerative disease in which there is both a movement disorder and dementia.

• The damage predominates in the basal ganglia.

• Increased protein carbonyl or oxidative damage to lipids or DNA.

Urinary 8-OHdG

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

• Detection by HPLC or ELISA

Biochemical pathways involved in the freeradical/oxidative stress theory of aging

Lipid peroxidation

• Measured lipid peroxidation by the thiobarbituric acid assay

• Thiobarbituric acid assay

– Reaction of aldhydic groups on products (e.g., malondialdehyde (MDA) and 4-hydroxy-2-nonenol (4-HNE)), which arose from free radical-initiated oxidative damage of polyunsaturated fatty acids.

Aging and oxidative stress

• Both long-lived and calorie-restricted animals constitutively have low levels of production of mitochondrial ROS, which could be responsible for their low rate of accumulation of mitochondrial DNA (mtDNA) mutations, and thus for their low rate of aging.

• Long-lived species have low degrees of fatty acid unsaturation (DBI, double bond index) in their cellular membranes, and thus lower levels of lipid peroxidation (MDA, malondialdehyde) and lipoxidation-derived protein modification (Prot. ox.).

Aging and oxidative stress

• The lower lipid peroxidation can also be partially responsible for the lower levels of oxidative damage in their mtDNA.

Mitochondrial theory of aging

• Increased ROS production

• Mitochondrial DNA (mtDNA) damage accumulation

• Progressive respiratory chain dysfunction

Protein Oxidation in aging, disease, and oxidative stress

• Attack of ROS on amino acids, generating oxo-, sulfo-, hydroxy-, chloro-, and nitro-derivatives

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

Protein Glycation

• Nonenzymatic reaction of sugars or of metabolites of sugars, amino acids, ascorbate, and lipids, with the free amine of a lysine or arginine residues

Lipid peroxidation products

• 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE)

• HNE

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.

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.