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

(s uperoxide radical) – H

O

(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 ca

n initiate another peroxidation reactio

n.

Intracellular sources of free radicals

• Mitochondrial electron transport system – Superoxide radical and semiquinone r

adical

• Microsomal (ER) electron transport syst em

– Superoxide radical and H

O

• Arachidonic acid metabolism

• Reactions within peroxisome

– Superoxide radical and H

O

Intracellular sources of free radicals

• In cytosol

– Xanthine oxidase oxidizes xanthine and generates H

O

– Amino acid oxidases generates H

O

as

their ordinary products

• H

O

and O

may diffuse from their subcel lular sites of production and affect the wh ole cell

• H

O

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 th

e generation of a disulfur bond between tw

o glutathione molecules to form oxidized gl

utathione

Dietary oxidants

• Generation of ROS

• ROS are reduced or inactivated through th

e generation of a disulfur bond between tw

o glutathione molecules to form oxidized gl

utathione

Xenobiotics

• Man-made compounds with chemical structure s 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 Fe

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)

• 2H

O

 2H

O ＋ O

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 sa

turate by H

O

at any concentration

Superoxide dismutase (SOD. EC 1.15.1.1 )

• Human SOD

– Cytosolic CuZn-SOD

– Mitochondrial SOD: MnSOD – Extracellular SOD

• 2O

＋ 2H

 H

O

＋ 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 ato m bridged by a common ligand: His 61

• Inactivation of copper- and zinc-containing

SOD by H

O

is the consequence of sever

al sequential reactions

Inactivation of cytosolic CuZn-SOD by H

O

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

O

• Oxidation of the Cu(I) by a second H

O

, t hus 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 c ontainig 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, lymp

h, and synovial fluid

EC-SOD

• Not induced by its substrate or other oxida nts (xanthine oxidase plus hypoxanthine, paraquat, pyrogallol, a-naphthoflavone, hy droquinone, catechol, Fe

, Cu

, buthionin e sulphoximine, diethylmaleate, t-butyl hy droperoxide, cumene hydroperoxide, sele nite, citiolone and high oxygen partial pres sure)

• Its regulation in mammalian tissues primar

ily occurs in a manner coordinated by cyto

kines, rather than as a response of individ

ual cells to oxidants

Nickel superoxide dismutase (Ni-SOD)

• Purified from the cytosolic fraction of Strep tomyces 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 ＋ H

O

2GSH GSSG

Glutathione peroxidase (GP)

• GP contains covalently bound Se (seleniu

m) 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 per oxidase GPX4 (PHGPX)

• Cytosolic GPX2 (GPX-G1)

• Extracellular GPX3 (or GPX-P)

• GPX5

– Expressed specifically in mouse epididymis, Sel

enium-independent

GPX

• cGPX and GPX1 reduce fatty acid hydrop eroxides and H

O

at the expense of GSH

• Cytosolic GPX2 (GPX-G1) and extracellul

ar GPX3 (GPX-P) are poorly detected in m

ost tissues except for the gastrointestinal tr

act and kidney, respectively.

GPX1

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

• The principal antioxidant enzyme for the d

etoxification of H

O

has for a long time be

en considered to be GPX, as catalase has

much lower affinity for H

O

than GPX

PHGPX

• Found in most tissues

• Highly expressed in renal epithelial cells and testes

• Located in both the cytosol and the membra ne fraction

• Directly reduce the phospholipid hydroperoxi

des, fatty acid hydroperoxides, and cholester

ol hydroperoxides that are produced in perox

idized membranes and oxidized lipoproteins

Tissue-specific functions of indivi dual glutathione peroxidases

• All glutathione peroxidases reduce hydrogen perox ide 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 (cG Px)

• Ubiquitously distributed

• Reduces only soluble hydroperoxides, suc

h as H

O

, and some organic hydroperoxid

es, such as hydroperoxyl fatty acids, cume

ne hydroperoxide, or t-butyl hydroperoxide

Gastrointestinal glutathione peroxidas es

(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 cG

Px

Plasma GPx (pGPx)

• Expressed in tissues in contact with body f

luids, e.g., kidney, ciliary body, and matern

al/fetal interfaces

Phospholipid hydroperoxide gluta thione peroxidases (PHGPx)

• Protects membrane lipids

• Reduces hydroperoxides of more complex lipids like phosphatidylcholine hydroperoxide

• Reduces hydroperoxo groups of thymine, lipopro teins, and cholesterol esters

• Unique in acting on hydroperoxides integrated in membranes

• Silence lipoxygenases

• Becomes an inactive structural component of the

mitochondrial capsule during sperm maturation

Glutathione reductase (GR)

glutathione reductase

GSSG ＋ H

 ^2GSH

NADPH NADP

Glucose-6-phosphate dehydrogena se (G6PD)

glucose-6-phosphate dehydrogenase, Mg

Glucose-6-phosphate  6-phosphoglucono-δ-lact one

DT-diaphorase

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

• In cytosol

• Two electron transfer of quinone compoun ds

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-tran

sferase (GST)

Heme oxygenase

• Heme  biliverdin bilirubin

• A major stress protein induced in cells res ponse to oxidant stress

• Bilirubin is an efficient plasma or serum sc

avenger of singlet

O

, O

, and peroxy rad

icals

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-media

ted signal transduction led to a proposal th

at ROS may be second messengers

Reactive oxygen species (ROS) as second messengers

• Generation of ROS by cytokines Ligand ROS

Tumor necrosis factor- H

O

/HO

Interleukin 1 H

O

/O

Transforming growth Factor-1 H

O

Platelet derived growth factor H

O

Insulin H

O

Angiotension II H

O

/O

Vitamin D

O

Parathyroid hormoneO

Oxidative stress and mitochondria

• During the course of normal oxidative pho

sphorylation, between 0.4 and 4% of all ox

ygen consumed is converted into the supe

roxide free radical (O

).

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 lucigen in

• Cytochrome c reduction

• Ferrous oxidation of xylenol orange

• 2’-7’-Dichlorodihydrofluorescence diacetat

e (DCFH-DA)

Chemiluminescence of luminol and luci genin

• Cell permeable method for ROS detection

• Luminol is sensitive to H

O

and peroxynitri te, but not sensitive to superoxide

• Lucigenin is specific for superoxide

Luminol-dependent CL assay

• The assay is based on the oxidation of lum inol by sodium hypochlorite (NaOCl). H

O

reacts with this oxidized product, generatin g an excited molecule capable of luminesc ence

• Specific for H

O

• Detect nM H

O

DCFH-DA

• DCFH-DA, a cell permeable, nonfluoresce nt precursor of DCF

• Intracellular esterases cleave DCFH-DA at the two ester bonds, produce a relatively p olar and cell-membrane imperable product , H

DCF

• H

DCF, can be oxidized by H

O

, yields th

e fluorescent DCF

DCFH-DA

^2,7- Dichlorodihydrofluorescein d iacetate (DCFH/DA)

• DCFH/DA diffuses through the cell membrane where it is enzymatically deacetylated by intra cellular esterases to the more hydrophilic nonf luorescent reduced dye dichlorofluorescein.

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

• DCF, excitated with 503 nm and emission at 5

23 nm.

DCFH/DA

• Hydroxyl radical, hydrogen peroxide and p erhaps a ferryl species, but not superoxide , may oxidize DCFH.

• The intracellular fluorescent measurement

s using dichlorofluorescein diacetate may r

eflect the ability of the test agent or toxican

t to generate hydroxyl radical.

DCFH/DA

• MW 487.3

• Dissolved in 50% methanol

• Did not dissolved in H

O or DMSO

Hydroethidium

• Measure superoxide anion concentration

• Superoxide anion can be measured by hy

droethidium oxidation into ethidium

Dihydroethidium

• Detect superoxide anion

Dihydroethidium Oxidation Ethidium

Blue fluorescent

Absorption/Emission 355/420 nm

Red fluorescent

Absorption/Emission

518/605 nm

O

production in electron transport chain

• Superoxide anions can be produced at bot h complex I and III

• Semiquinone formation at both complex I

and III results in the production of superoxi

de 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 R OS, mainly through the production of supero xide anion

• The two major sites of superoxide production

are at complex I and complex III

Sites of superoxide formation in the respirat

ory chain

Superoxide production in mitochondria

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

n’

• At complex III (bc1 complex)

– was cytochrome b rather than ubisemiquino

ne

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 o

f accumulation of mitochondrial DNA (mtDN

A) mutations, and thus for their low rate of agi

ng.

Aging and oxidative stress in mammals and birds

• Long-lived species also have low degrees of fa

tty acid unsaturation (DBI, double bond index)

in their cellular membranes, and thus lower lev

els of lipid peroxidation (MDA, malondialdeh

yde) and lipoxidation-derived protein modifica

tion (Prot. ox.). This lower lipid peroxidation c

an also be partially responsible for the lower le

vels of oxidative damage in their mtDNA.

Mitochondrial DNA

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

• mtDNA, unlike nuclear DNA, is not protecte

d 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-add

uct radicals and allyl radical of thymine

Product formation from the C5- and C6-OH-add

uct 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-O

H-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 co

ntrol subjects.

Breast cancer

• Significantly higher (P < 0.0001) levels of 8-O

H-dG in DNA from tumour, compared non-tu

mour tissue

Cervical cancer

• Levels of 8-OH-dG significantly increased (P

< 0.001) in DNA from low-grade and high-gra

de levels of dysplasia, compared to normal, alt

hough this did not correlate with human papill

omavirus status.

Oxidative DNA damage measurements in non-cancerous pathological conditions

• Parkinson’s disease (PD)

– DNA levels of 8-OH-dG significantly elevat ed (P = 0.0002) in substantia nigra of PD br ains

• Alzheimer’s disease

– Higher levels of 8-OH-dG in cortex and cere

bellum 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 high er in SLE patients vs.controls (P = 0.0001) – Titres of serum autoantibodies to 5-OHMeU

ra 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 chromo some, prevent chromosome fusion and genomi c instability.

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

• Telomeres shorten with cell division.

Senescence is due to downregulation

of positive-acting cell cycle regulatory genes

• c-fos proto-oncogene

• Genes for Cdc2 and cyclin A and E, componen ts 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 associate

d with permanent loss of protein function and

may lead to the degradation of the damaged pr

oteins by proteasome and other proteases or to

their progressive accumulation.

Oxidative/nitrosative modifications of prote in Cys residues

• ROS/RNS may induce the formation of mixed disulphide s between protein thiol groups (PSH) and GSH to form S-glutathionylated proteins (PSSG).

– PSH may be initially “activated” by oxidative/nitrosative modi fications to give thyil radical (PS·), sulphenic acid (PSOH), or protein S-nitrosothiol/S-nitrosated protein (PSNO). These mod ifications may be either stabilized as such or react with GSH t o the mixed disulphide (PSSG). All these modifications are re versible and can be reduced back by increases in the GSH/GS SG ratio, reduced thiols, or enzymatic reactions. Otherwise,

• PSSG may be generated by thiol/disulphide exchange rea

ction with GSSG or by reaction with other “reactive” inte

rmediates of GSH, such as GSNO.

Oxidative/nitrosative modifications of prote in Cys residues

• PSOH may also be irreversibly oxidised by ROS/

RNS to form sulphinic (PSO2H)

• and sulphonic (PSO3H) derivatives, leading to irr eversible loss of biological activity. PSH may als o be oxidised to

• disulphide both within and between proteins (PS

SP). PSSP can be reversed by enzymes (protein

disulphide isomerase and thioredoxin/thioredoxi

n reductase) or reducing agents.

Methionine sulfoxide reductases

• Moskovitz, J.

Biochimica et Biophysica Acta 1703: 213– 219 (2 005)

• Enzymes involved in antioxidant defense, protein r egulation, and prevention of aging-associated dise ases

• Met oxidation may play an important role in the de

velopment and progression of neurodegenerative d

iseases like Alzheimer’s and Parkinson’s diseases.

Methionine and cysteine

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

• Cysteine can be regenerated by a number of non-enzy matic (e.g. glutathione) and enzymatic pathways (e.g.

involving NADPH-dependent enzymatic reactions)

• For MetO reduction an addition of Msr enzymes is ne

eded.

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 o xidation and reduction

• Potassium channel of the brain

• Calmodulin

• Reversal methionine oxidation may play an im

portant role in regulation of protein’s function

either directly or mediated by signal transducti

on 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 synchro nize internal rhythms by the periodic release of melatonin.

• Melatonin exerts immunoenhancing action, bo

th in animals and in humans.

Significance of melatonin secretion decline

for reduced antioxidant protection in elderly

Melatonin

• A potent free radical scavenger and antioxidan t that scavenges especially highly toxic hydrox yl radicals

• Stimulates a number of antioxidative enzymes

• Melatonin is both lipophylic and hydrophilic a

nd diffuses widely into cellular compartments,

thus providing on-site protection against free r

adical 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 th e pathogenesis of neurodegenerative diseases c haracteristic of aged population.

• Neurodegenerative diseases such as Alzheimer

’s and Parkinson’s because of high vulnerabilit

y of the central nervous system to oxidative att

ack and neoplastic disease.

Alzheimer’s disease

• Features

– Amyloid- plaques

– Neurofibrillary tangles, and extensive neural l oss, particularly in the hippocampus and cere bral 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 survi val.

• Decreased melatonin concentrations were obse

rved in some, but not all, patients suffering fro

m Alzheimer’s disease.

Parkinson’s disease

• Features

– Progressive deterioration of dopamine-conta ining neurons in the pars compacta of the su bstantia nigra in the brain stem.

• The loss of these neurons is caused by auto-oxi

dation of dopamine due to relatively high expo

sure of these neurons to free radicals.

Parkinson’s disease and melatonin

• In experimental animal models of Parkinson’s

disease, melatonin administration diminished l

ipid peroxidation that occurred in the striatum,

hippocampus and midbrain after injection of 1-

methyl-4-phenyl-1,2,3,4-tetrahydropyridine an

d reduced cytotoxicity of 6-hydroxydopamine

Consequences of ROS/RNS and oxidative/

nitrosative stress on protein function and fat

• ROS/RNS may cause oxidative/nitrosative mo e

difications on sensitive target proteins.

• Reversible modifications, usually at Cys and

Met residues, may have a dual role of modulati

on of protein function and protection from irre

versible modification.

Oxidatively modified proteins in agi ng and disease

Protein oxidation

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

• Direct oxidation of protein side chains

– Oxidation of the side chains of lysine, prolin e, 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 o xidation

– Reaction of protein carbonyls with 2,4-dinitr ophenylhydrazine (DNPH) to form the corre sponding hydrazone

– The levels of the protein carbonyl levels are

measured by the absorbance of the 2,4-dinitr

ophenylhydrazone at 370 nm

Measurement of 3-nitrotyrosine

• By using HPLC with the electrochemical dete ction

• 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 hep atocytes, 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 s pan, and this correlates with reduced protein ca rbonyls.

• Transgenic mice with a knockout of methionin

e sulfoxide reductase, which repairs oxidized

methione, have a reduced life span and show i

ncreased protein carbonyls.

Proteins vulnerable to oxidative damage

• Not all proteins are uniformly susceptible to o xidative damage.

• Mitochondrial aconitase was particularly vulne rable to oxidative damage accompanying agin g in drosophila.

• Mitochondrial adenine nucleotide translocase,

glutamine synthetase and creatine kinase are p

articularly vulnerable to oxidative damage.

Alzheimer’s disease

• Neuropathologic hallmarks are senile plaques contain ing -amyloid and neurofibrillary tangles, which occu r in pyramidal neurons of the cerebral cortex and hipp ocampus.

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

• Protein carbonyls were significantly increased in both

hippocampus and the inferior parietal lobule, but unc

hanged in the cerebellum, consistent with the regional

pattern of histopathology in AD.

Alzheimer’s disease

• Significant decreases in glutamine synthetase a nd creatine kinase activity.

• Oxidative damage to the glial glutamate transp orter

• Increases in protein carbonyls both in neurofi

brillary tangles as well as in the cytoplasm of t

angle 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 c

ytoplasmic inclusions in the substantia nigra n

eurons known as Lewy bodies.

Parkinson’s disease

• Increases in protein carbonyls in all brain r egions including the substantia nigra, basa l ganglia, globus pallidus, substantia inno minata, 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 neurodegene rative disease in which there is both a moveme nt disorder and dementia.

• The damage predominates in the basal ganglia.

• Increased protein carbonyl or oxidative damag

e 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 free

radical/oxidative stress theory of aging

Lipid peroxidation

• Measured lipid peroxidation by the thiobarbitu ric 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 polyun

saturated fatty acids.

Aging and oxidative stress

• Both long-lived and calorie-restricted animals co nstitutively have low levels of production of mito chondrial ROS, which could be responsible for th eir low rate of accumulation of mitochondrial DN A (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 ce

llular membranes, and thus lower levels of lipid p

eroxidation (MDA, malondialdehyde) and lipoxid

ation-derived protein modification (Prot. ox.).

Aging and oxidative stress

• The lower lipid peroxidation can also be partia

lly responsible for the lower levels of oxidativ

e damage in their mtDNA.

Mitochondrial theory of aging

• Increased ROS production

• Mitochondrial DNA (mtDNA) damage accum ulation

• 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-de rivatives

• Oxidative attack of polypeptide backbone i

s initiated by the

OH-dependent abstractio

n 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 met

abolites of sugars, amino acids, ascorbate,

and lipids, with the free amine of a lysine o

r 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 mitochondri al DNA is inversely correlated with maximum life span in the heart and brain of mammals.

This inverse relationship is restricted to mtD

NA, 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 F3 44 rats, male B6D2F1 mice, male C57BL/6 mice , and female C57BL/6 mice) of various ages wer e 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 n DNA with age in all tissues and strains of rodents studied.

• Age-related increase in oxo8dG in mtDNA i

solated from the livers of the rats and mice.