Transcript 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
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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
subcellular 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
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Catalase
Superoxide dismutase
Glutathione peroxidase
Glutathione reductase
Gluththione S-transferase
Glucose-6-phosphate dehydrogenase
DT-diaphorase
Catalase (EC 1.11.1.6)

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 H O at any concentration
• 2H2O2
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Superoxide dismutase
(SOD. EC 1.15.1.1)
• Human SOD
– Cytosolic CuZn-SOD
– Mitochondrial SOD: MnSOD
– Extracellular SOD
• 2O2-. ＋ 2H＋
 H 2O 2 ＋ 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
majority 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, anaphthoflavone, 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
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,
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
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
and testes
• Located in both the cytosol and the
membrane fraction
• Directly reduce the phospholipid
hydroperoxides, 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
hydroperoxides, such as hydroperoxyl
fatty acids, cumene hydroperoxide, or tbutyl 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
lipids like phosphatidylcholine hydroperoxide
• Reduces hydroperoxo groups of thymine,
lipoproteins, 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＋

NADPH
2GSH
NADP＋
Glucose-6-phosphate
dehydrogenase (G6PD)
glucose-6-phosphate dehydrogenase, Mg2+
Glucose-6-phosphate  6-phosphoglucono-δlactone
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 Stransferase (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 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
Tumor necrosis factor-
Interleukin 1
Transforming growth Factor-1
Platelet derived growth factor
Insulin
Angiotension II
Vitamin D3
Parathyroid hormone
ROS
H2O2/HO
H2O2/O2-
H2O2
H2O2
H2O2
H2O2/O2-
O2-
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
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
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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
Oxidation
Ethidium
Blue fluorescent
Red fluorescent
Absorption/Emission
Absorption/Emission
355/420 nm
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
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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
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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-OHadduct radicals and allyl radical of thymine
Product formation from the C5- and C6-OHadduct radicals and allyl radical of thymine
Product formation from the C5- and C6OH-adduct radicals of cytosine in the
presence of oxygen
Product formation from the C5- and C6OH-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, 8OH-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 8OH-dG in DNA from tumour, compared nontumour tissue
Cervical cancer
• Levels of 8-OH-dG significantly increased (P
< 0.001) in DNA from low-grade and highgrade 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 5OHMeUra 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 downregulation
of 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 fate
• 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 modifications to give thyil
radical (PS·), sulphenic acid (PSOH), or protein Snitrosothiol/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
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 agingassociated 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 nonenzymatic (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 dopaminecontaining neurons in the pars compacta of
the substantia nigra in the brain stem.
• The loss of these neurons is caused by autooxidation 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 1methyl-4-phenyl-1,2,3,4-tetrahydropyridine
and reduced cytotoxicity of 6hydroxydopamine
Consequences of ROS/RNS and
oxidative/nitrosative stress on protein
function and fate
• ROS/RNS may cause oxidative/nitrosative
modifications 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 4hydroxynonenal, a product of lipid peroxidation
Measurement of protein carbonyls
• The most widely utilized measure of protein
oxidation
– Reaction of protein carbonyls with 2,4dinitrophenylhydrazine (DNPH) to form the
corresponding hydrazone
– The levels of the protein carbonyl levels are
measured by the absorbance of the 2,4dinitrophenylhydrazone 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 antiinflammatory 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 free
radical/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 4hydroxy-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 nitroderivatives
• Oxidative attack of polypeptide backbone
is initiated by the OH-dependent
abstraction of the -hydrogen atom of an
amino acid residue to form a carboncentered 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 4hydroxyhexenal (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-dihydro2’-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.