The Price for Living:

Free Radical Generation and

Defense Mechanism

Free Radicals
vs
Anti Oksidan
Noor Wijayahadi
 Bacteria Anti-biotic

Free Radicals Anti-oxidants

(Oxidants)
 RECORDS IN LIFE SPAN

Kura-kura 170 tahun Kucing 30 tahun

Manusia 116 tahun Anjing 27 tahun
Kerang 80 tahun Sapi 20 tahun
Kakatua 70 tahun Kelinci 18 tahun
Gajah 70 tahun Ayam 14 tahun
Burung hantu 68 tahun Tikus 5 tahun
Kuda 62 tahun Mencit 5 tahun
Chimpanse 50 tahun Coro 1 tahun
Gorilla 48 tahun Nyamuk 5 bulan
Beruang 47 tahun Lalat 70 hari
Merpati 30 tahun
THEORIES OF AGING PROCESS :

Genetic Clock Theory

Somatic Mutation Theory
Rate of Living or Metabolism Theory
Immune System Destruction Theory
Free Radicals Theory
Lain-Lain:
kesepian, banyak pacar, banyak hutang/kredit, banyak
pasien, dll
 What are free radicals?
Adalah molekul yang kelebihan elektron
 sangat reaktif

1.Reactive Oxygen Species (ROS)

2.Reactive Nitrogen Species (RNS) - NO
3.Reactive Metabolites or Intermediates
 produk metabolisme obat, toxin, pollutant,
merokok, dll
.. — . .
¨ .. ¨ .
. O—O — .
—
N    O
¨ ¨
Molecular Oxygen
Nitric Oxide
(a radical)
(a diradical)
Origins of reactive oxygen, nitrogen
and chlorinating species
Functions of oxidant
 1. Reactive Oxygen Species
(ROS)
• Superoxide (O2.-)
• Hydrogen Peroxide (H2O2)
• Hydroxyl Radical (OH.) - product of Fenton
reaction catalyzed by free Fe and Cu
• Singlet Oxygen (1gO2) - oxygen at an excited
state, requiring photosensitizers and photons
 Sumber “oxygen free radicals”

• In mitochondria:
- generation of energy - ATP
- glucose, fatty acids, amino acids
- O2 2H2O
4e-
- leakage of O2-. (superoxide)
H2O2 (hydrogen peroxide)
• In Smooth Endoplasmic Reticulum
(microsome)
- detoxification (cytochrome P-450s)
- toxins, drugs and xenobiotics
- O2 + RH R-OH and H2O
- leakage of O2-.
- metabolic activation - X.
• In Smooth Endoplasmic Reticulum
(microsome)
- eicosanoids synthesis
- substrate: arachidonic acid (AA)
- O2 + AAs prostaglandins,
thromboxanes and leukotrienes
- leakage of O2-.
• In Peroxisomes
- containing oxidases for degradation of
various substrates
- glucose, amino acids, xanthine, etc.
- requires O2
- byproduct is H2O2
• In Cytoplasm
- nitric oxide (NO.) production from
Arginine
- functions as a biological messenger
- in brain, vascular endothelial cells, and
macrophages
- NO. + O2-. ONOO. (peroxynitrite)
 2. NO: a Biological Messenger

• NO is a neurotransmitter (brain- bNOS)

• NO regulates blood pressure (vascular
endothelial cells- eNOS)
• NO is a cytotoxic agent (macrophages- iNOS)
 3.1. Sources of Superoxide Anion

• Enzymic reactions • Environmental factors

NADH oxidase ultraviolet light
NADPH-P450 reductase X-rays
xanthine oxidase toxic chemicals
aromatic hydroxylamines
• Cellular sources aromatic nitro compounds
leukocytes and macrophages insecticides
mitochondrial electron transfer chemotherapeutic agents
microsomal monooxygenase

1341
 3.2. Sources of Hydrogen Peroxide

• Enzymic generation • Dismutation of O2.–

(non radical) (radical)
glycolate oxidase
acetyl-CoA oxidase O2.– + O2.– + 2H+  H2O2 + O2
D-amino acid oxidase
NADH oxidase
urate oxidase
monoamine oxidase

1342
 3.3. Sources of Hydroxyl Radical
Most of the HO. generated in vivooriginates from the
breakdown of H2O2 via a Fenton reaction

The Fenton reaction entails a transition metal-dependent reduction of H 2O2 to HO.

Fe++ + H2O2 Fe+++ + HO– + HO.

The reaction requires a reduced transition metal, process accomplished by O2.–

Fe+++ + O2.–  Fe++ + O2

The overall reaction, involving iron reduction by O2.– and iron oxidation by H2O2

Fe+++ + O2.–  Fe++ + O2

Fe++ + H2O2 Fe+++ + HO– + HO.
1343
 3.3. Sources of Nitric Oxide
Nitric oxide is generated by a Ca-dependent
ed function oxidase —nitric oxide synthase (NOS
NH
2
in mammalianNHtissues2
| |
C=NH2 C=O
| |
NH NH
| |
CH2)3 + O2 + NADPH CH2)3 + NO + NADP+
.
|
CH–COO– NOS |
CH–COO–
| |
NH3 NH3

1343
 3.3. Sources of Nitric Oxide
Nitric Oxide Synthases (NOS) in mammalian tissues

arginine + O2 + NADPH citrulline + .NO + NADP+

NOS

NOS-I NOS-II NOS-III

Neuronal Macrophage Endothelial
Constitutive inducible Constitutive
Ca2+ Ca2+

1343
 Types of NOS
• NOS I
– Central and peripheral neuronal cells
– Ca+2 dependent, used for neuronal communication
• NOS II
– Most nucleated cells, particularly macrophages
– Independent of intracellular Ca+2
– Inducible in presence of inflammatory cytokines
• NOS III
– Vascular endothelial cells
– Ca+2 dependent
– Vascular regulation
 3.3. Sources of Nitric Oxide
Nitric Oxide Synthases (NOS) in mammalian tissues
Mayor Domains

arg calmodulin NADPH

FMN
Heme FAD
BHT

oxygenase domain reductase domain

arginine + O2 + NADPH citrulline + .NO + NADP+

1343
 Oxygen Metabolism
O2

RBC Hb-O2

Cells

PS NOS Membrane Peroxisome SER Mitochondria

h e - transport
Oxidases PHS P-450
MPx O2 O2
O2 AA O2
NO.
H2O2 PGs
NO.
H2O O2.-
H O O .-
O2
1

O
. -
HOCl- H2O2 LTs
2 2
2
 Oxygen Consumption and
Oxidative Stress

• O2 Consumption: 4 x 1010 O2/cell/hr

• H2O2 Production: 3.24 x 109 H2O2/cell/hr
Mitochondria - 13.6%
Microsomes - 47.7%
Peroxisomes - 34.1%
Cytosol - 4.6%

Boverls, et al. Biochem. J. 128:617 (1972) where rat liver at 22 0C was used
 Oxidative Damage
Free Radicals

Proteins Lipids DNA/RNA

(-SH) (R-OO.) (-OH.)
 Severity of Oxidative Stress &
Biological Consequences
Severity of Biological
Oxidative Stress Consequences

A. Low level & gradual Aging

Carcinogenesis
B. Medium level & rapid Mutagenesis

C. Large level & rapid Death, Stroke,

Trauma, Ionizing
irradiation
 Free Radical Involvement
in Pathophysiological Conditions OXIDANTS ANTIOXIDANTS

 Adriamycin cardiotoxicity  Exercise

 AIDS  Favism
 Adult Resp Distress Synd  Iron Overload
 Aging  Myocardial Infarction
 Alcoholism  Oxygen Toxicity
 Alzheimer’s Disease  Parkinson’s Disease
 Amyotrophic Lateral  Radiation Therapy
Sclerosis  Smoking
 Atherosclerosis  Stroke
 Diabetes  Trauma
 Cancer
Breathing is bad for you
• All breathing generates oxygen radicals, which
are the main sources of mutation in DNA, leading
to cancer, birth defects, and various peculiarly
shaped molecules in urine.
• Breathing has been observed three minutes
before death in 100% of fatalities.
• We urge everyone to stop breathing until
further research has been carried
out.
 Oxidative Stress

OXIDANTS ANTIOXIDANTS

Oxygen Free Radicals Specific Enzymes
Nitrogen Free Radicals
Vitamin E, Vitamin C,
Carotenoids, Selenium
 Free Radical Defense System
(i) preventative mechanisms
(ii) repair mechanisms
(iii) physical defences
(iv) antioxidant defences

• Antioxidant Enzymes
• Antioxidant Quenchers
• Antioxidant from Foods –
nutrients/non-nutrients
 Antioxidants

• Prevents the transfer of electron from O 2 to

organic molecules
• Stabilizes free radicals
• Terminates free radical reactions
Antioxydant defence
mechanisms
 Primary Antioxidant Defenses
Specific Antioxidant Small Antioxidant
Enzymes Molecules
 Superoxide dismutase  Vitamin E
 Catalase  Vitamin C
 Glutathione Peroxidases  Coenzyme Q
 Glutathione Transferases  Uric Acid
 Carotenoids

OXIDANTS ANTIOXIDANTS

1346
Antioxydant network to maintain redox balance
 Antioxidant Enzymes

GSH
CuZnSOD Peroxidase

O2•¯ H2O2 H2 O + O 2
Fe2+
Mn SOD Catalase

OH•
 Antioxidant Enzymes-1
• Superoxide Dismutase (SOD) – to get rid of
superoxide produced from electron
transport chain, the product is hydrogen
peroxide.
– MnSOD (mitochondria).
– CuZn SOD (cytosol).
 Antioxidant Enzymes - 2
• Glutathione Peroxidase (GSH PX) – to get
rid of hydrogen peroxide (H2O2) and some
lipid peroxide. It requires reduced
glutathione (GSH) as substrate and
produces oxidized glutathione (GSSG) as
product. A cytosolic enzyme.

Antioxidant Enzymes - 3
• Catalase –to get rid of hydrogen peroxide
produced in peroxisome.
 Glutathione Synthesis
Protein
Methionine
Cysteine
Glutamate
 -Glutamylcysteine
Glycine
 Supplementation of
Cysteine Prodrugs
Protein Supplementation:

Methionine OTC
NAC
Cysteine
MET
Glutamate
GSH
-Glutamylcysteine
Glycine
 Cysteine Prodrugs
• NAC – N-Acetyl-Cysteine
• OTC - L-2-oxothiazolidine- 4- carboxylate
• GSH – Glutathione
• MET – Methionine analogs
• others
 Conversion of OTC to Cysteine by
Oxoprolinase
S
H Oxoprolinase S CH2
O
N CO -
H O ATP
ADP + Pi - -
COO CHCOO
NH3
L-2-oxothiazolidine-
4- carboxylate
HS CH2 CO2 Carboxy- L-
(OTC)
cysteine
-
CHCOO
NH3
L- Cysteine
 Antioxidant Quenchers
• Cellular proteins which chelate pro-oxidant
minerals (iron and copper or others)
• Transferrin – iron transport protein
• Ferritin – iron storage protein
• Metallothionein – minerals and heavy
metals (Zn/Cu/Cd/Hg)
• Ceruloplasmin – copper transport and
storage
 Antioxidants From Food
• Antioxidant nutrients
– vitamin E
– vitamin C
– (vitamin A?)
– beta-carotene
• Phytochemicals – antioxidants from
plants
 Food
Nutrients Non-nutrients

Energy, building Factors regulating

materials metabolism Phyto-
chemicals
•pigments
•Antioxidants
Lipid Protein Carbo- Water Vitamins Minerals
hydrate Fibers

Zn, Se Other food

EFA & EAA & C, E,  -Car
components
non-EFA non-EAA Glucose
Functional food
or Neutraceuticals
Cys GSH
 Discovery of Other Functions
of Phytochemicals

• Anti-oxidant • Anti-cholesterolemic
• Anti-inflammatory • Anti-hemorrhagic
• Anti-estrogenic • Anti-mutagenic
• Anti-allergic • Anti-neoplastic
 Antioxidant Network
GSH

Vitamin E GSSG
Phytochemicals

Vitamin E
radicals Vitamin C Phytochemicals-O

Vitamin C
Radicals
 Effect of Oxidative Stress on
Oxidative Stress Gene Expression (NF B Activation)

Oxidative Stress

NF-B Activation

Gene Transcription
Cytokines, Chemotactic Factors

Inflammation

ROS

Oxidative
Damage
 Effect of antioxidants on
the inhibition of NFB activation

Oxidative Stress

NF-B Activation

Gene Transcription
Antioxidants Cytokines, Chemotactic Factors
vitamin E

Inflammation

ROS

Oxidative
Damage
 Prevention of Free Radical-Mediated
Chronic Diseases
Genetic Environmental Viral Dietary

Developmental ROS Hormonal

Anti-
NF-B Activation Inflammatory

Gene Transcription
Amplification Cytokines, Chemotactic Factors

Inflammation

ROS Antioxidants

Cell Death
Inflammation is a source of PROSTATE
HEALTHY
endogenous mutagenic chemicals PROSTATE
CANCER
TISSUE

ONOO- Nitrations

NO.

ANTIOXIDANTS
HO. Oxidations DNA
iNOS ANTI-INFLAMMATORY
Fe AGENTS 2+
damage

H2O
PHAGOCYTE
NADPH O2.- Nox (mitogenic
Oxidase
oxidase)
O2 MPO
2
EPO

HOCl
tro phil Halogenations
neu
5 % in
prot
e HOBr
BIOMARKER KERUSAKAN OKSIDATIF
Hidup = Keseimbangan
Pembentukan Radikal-Bebas vs
Mekanisme Pertahanan