Oxidants and their role in CVS

• Oxidation is the loss of electron or an increase in oxidation state by a molecule atom or ion.

• Reduction is the gain of electron or a decrease in oxidation state by a molecule atom or ion.

• In chemistry, a radical (more precisely, a free radical) is an atom, molecule or ion that has unpaired valence electrons.

Oxidative stress

It has been implicated in the ageing process & in many diseases (e.g., atherosclerosis and coronary heart diseases).

Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage.

Oxidation is a chemical reaction involving the loss of electrons or an increase in oxidation state. Oxidation reactions can produce free radicals. These radicals can start chain Reactions. Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen. Examples : peroxides, superoxide, hydroxyl radical, and singlet oxygen.

What are free radicals?

• Any molecule containing one

or more unpaired electrons.

• These unpaired electrons

readily form free radical

molecules which are

chemically reactive and

highly unstable.

How are ROS Formed?

• Primary source is our body during energy production

• Environmental contaminants

• Ionizing and ultraviolet radiation

• Prolonged high blood flow states (atherosclerosis, heart attacks and stroke)

• Diet (fatty and processed foods)

• Low levels of antioxidants

Types of free radicals

1. Superoxide, O2-

2. Hydrogen peroxide, H2O2

3. Hydroxyl radical, OH-

4. Singlet oxygen, 1O2

5. Hydroperoxy radical, HOO-

6. Lipid peroxide radical, ROO-

7. Nitric oxide, NO-

8. peroxynitrite, ONOO-

Responsible for more than 70% of deaths

Cancer

Heart Diseases

Alzheimer's disease Ageing

Atherosclerosis

CVA (Stroke)

Free radical-Mediated Diseases

• Diseases of the old -

Chronic and degenerative diseases

(diabetes, cataracts, Alzheimer’s disease, cancers,

cardiovascular disease, and aging)

• Diseases of the young and innocent - Acute and immature diseases

(Eyes: retinopathy of prematurity, Lung: bronchopulmonary displasia, Brain: cerebral pulsy, Pancreas: Type 1 diabetes)

Cells are attacked and damaged by Free Radicals

Free radicals are highly reactive molecular species

• Free radicals are highly reactive molecular species with an unpaired electron.

• They can react with, and modify, proteins, nucleic acids and fatty acids in cell membranes and plasma lipoproteins.

Cells are Damaged by Free Radicals

• Free radical goes around the cell literally stealing electrons from healthy molecules.

• Free radicals Cause damage to: – Cell membranes (Lipids). – Enzymes and other Proteins. – Plasma lipoprotein. – Chromosomes and DNA.

Free Radicals are Formed in the Body Under Normal Conditions

• In eukaryotic organisms, Free Radicals are mainly generated during the normal metabolism such as: – Cellular Respiration

– Process involving:

• Oxygen

• Oxidases

• Electron transports in mitochondria or endoplasmic reticulum.

Several powerful oxidants (Reactive Oxygen Species) are produced

during the course of metabolism • Superoxide. • Hydrogen peroxide (H2O2) • Peroxyl radicals (ROO) • Hydroxyl radicals (OH) • Lipid oxyl • Singlet oxygen • Peroxinitrite formed from nitrogen oxide

(NO)

Superoxide is an anion (O2−)

with one unpaired electron

• Superoxide is formed when oxygen takes up one electron.

• It is the product of the one-electron reduction of dioxygen O2.

• Superoxide ion is a free radical generated during variety of metabolic reactions.

Superoxide is by-product of various reactions

• Superoxide is formed in RBC by the auto-oxidation of hemoglobin to met-hemoglobin. – Approximately 3% of hemoglobin in

human RBCs auto-oxidize per day.

Fe+++ Fe++

Neutrophils exhibit a respiratory burst and produce ROS

• The attachment of microorganisms to phagocyte membrane initiates the process of phagocytosis and respiratory burst.

• The "respiratory burst" describes a metabolic pathway (Hexose Monophosphate Shunt), that produces superoxide anion, singlet oxygen, hydroxyl ion and hydrogen peroxide.

Superoxide is used in oxygen-dependent killing mechanisms of microorganisms

• The reaction catalyzed by NADPH oxidase.

Phagocyte

Bacteria

Cells engulfs a solid particle to form an internal vesicle known as phagosome.

Xanthine oxidase (liver, involve in proteolytic modification) generates (ROS)

During severe liver damage, xanthine oxidase is released into the blood, so a blood assay for XO is a way to determine if liver damage has happened.

The use of oxygen as part of ETC for generating ATP produces ROS

• During electron transport through respiratory chain, the superoxide anion is produced as a by-product of several steps.

• Particularly important is the reduction of coenzyme Q in complex III, since a highly reactive free radical is formed as an intermediate (Q·−).

Mitochondrial ROS generation is the major source of oxidative stress

ROS ROS

Superoxide is generated when electrons jump directly to oxygen instead of moving

through the normal ETC

Cytochrome-P450 mediated reactions are source of electrons and

therefore generate ROS • Cytochrome P450 enzymes are present in

most cells of the body (Inner membrane of mitochondria and in the endoplasmic reticulum).

• Play important roles in variety of metabolic reactions.

Cytochrome-P450 Play important roles in variety of metabolic reactions • Hormone synthesis and breakdown.

• Cholesterol synthesis

• Vitamin D metabolism.

• Metabolize potentially toxic compounds: – Drugs

– Products of metabolism such as bilirubin.

C

CH3

=O

ROS is generated as intermediate in reactions catalyzed by

P450 enzyme system

Formation of Pregnelonone from cholesterol is an example of electron transport reaction

catalyzed by P450

H C H2

C H2

C H2

C H

CH3

CH3

The Hydroxyl radical (•OH) is unstable

• Reacts rapidly and non-specifically with most biological molecules.

• Can damage cells by: – Initiating reactions such as lipid peroxidation:

• Damage to cell membrane. • Changes in LDL.

– Oxidation of DNA: • Mutations and possibly cancer.

– Oxidation of Proteins: • Enzyme inhibition, denaturation and protein degradation.

•OH (hydroxyl radicals) are generated by Haber-Weiss reaction

• The Haber-Weiss reaction generates •OH (hydroxyl radicals) from H2O2 (hydrogen peroxide) and superoxide (•O2-).

• This reaction can occur in cells and is therefore a possible source for oxidative stress.

• The reaction is catalyzed by iron.

Haber-Weiss reaction is an iron catalyzed two step mechanism that generates •OH (hydroxyl radicals)

• The first step of the catalytic cycle involves reduction of ferric ion to ferrous:

• Fe+3 + •O2− → Fe+2 + O2

• The second step is the Fenton reaction: • Fe+2 + H2O2 → Fe+3 + OH− + •OH

• Net reaction:

• •O2- + H2O2 → •OH + OH- + O2

• Haber-Weiss reaction is one of the mechanisms involved in tissue injury of hemochromatosis

Metabolism of PUFA (Lipid peroxidation) is a source of free radicals

• This process proceeds by a free radical chain reaction mechanism.

• In this process fatty acid radicals are generated.

• Fatty acid radicals damage the lipids in cell membranes, resulting in cell damage.

Lipid peroxidation Lipid peroxidation refers to the

oxidative degradation of lipids.

It is the process whereby free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage.

Process proceeds by a free radical chain reaction mechanism. It most often affects polyunsaturated fatty acids(PUFA).

end products of lipid peroxidation may be mutagenic and carcinogenic

• In Cytoplasm

- nitric oxide (NO.) production from Arginine

- functions as a biological messenger

- in brain, vascular endothelial cells, and macrophages

- NO. + O2-. ONOO. (peroxynitrite)

Peroxynitrite seems to be the major player in Atheroma formation

• Superoxide anion (O2-) reacts quickly with nitric oxide (NO) in the vasculature.

• The reaction produces peroxynitrite and depletes the bioactivity of NO.

• This is important because NO is a key mediator in many important vascular functions including: – Regulation of smooth muscle tone and blood

pressure. – Platelet activation.

Free Radical damage is an important factor in development of Atheroma

• ROS attacks the LDL.

• LDL are modified and loose their identity so that can not be recognized by macrophages.

• Macrophages react against LDL.

• The resulting inflammatory reaction is the cause of Atheroma development.

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)

• Production of Singlet Oxygen

- Photosensitizers in the biological system

(bilirubin, riboflavin, retinal, porphyrin)

- requires light , O2 and photosensitizers

- chlorophyll in photosynthesis

- photodynamic therapy

Generation of Free Radical

Environmental effects: a) due to drug metabolism.

b) due to damages caused by UV or X-rays

c) cigarette or alcohol.

FREE RADICAL FORMATION FREE RADICALS : THE CAUSE OF

VIRTUALLY ALL DISEASES

Industrial pollution

Pesticides & herbicides

High fat foods

Environmental pollution

Excessive Alcohol & smoking

Harmful effects of free radicals

A. Free Radical and biomolecules

1. Proteins

Cause oxidation of sulfhydryl groups, and modification

of AA. ROS may damage protein by fragmentation,

aggregation results in the loss of biological activity

of proteins. 2. Lipids

The polyunsaturated lipid molecules of cell membranes

are particularly susceptible to damaging free

radicals process and contribute to the uncontrolled

chain reaction (lipid peroxidation).

Harmful effects of free radicals

A. Free Radical and biomolecules

3. Carbohydrates

Glycation increases the susceptibility of proteins to

the attack by free radicals.

4. Nucleic acid

cause DNA strand breaks, fragmentation of bases

and deoxyribose results in cytotoxicity and

mutations.

Harmful effects of free radicals

B. Diseases

1. Cardiovascular diseases (CVD): ox-LDL, formed by the action

of free radicals, promote CVD and atherosclerosis (AS).

2. Cancers: damage DNA and cause mutation and

cytotoxicity, play a key role in carcinogenesis.

3. Inflammatory diseases: damage on the extracellular

components such as collagen and hyaluronic acid,

promote glomerulonephritis and ulcerative colitis.

4. Respiratory diseases: destroy endothelium and cause lung

edema. Cigarette smoke contains free radicals and

promotes the production of more free radicals.

Macrophage take up oxidized LDL, when

overload with lipid, become “foam cells”.

Conglomerate of foam cells form fatty streaks

or yellow patches visible in the arterial

wall.

Dying foam cells release lipid that form lipid

pool within the arterial wall.

Derived Oxidants to Convert LDL into an

Atherogenic Form

Atherosclerosis may be due to free radical reactions involving diet-derived lipids in the arterial wall and serum to yield peroxides and other substances. These compounds induce endothelial cell injury and produce changes in the arterial walls .

Athersclerotic plaque Formation

Harmful effects of free radicals

B. Free Radical and diseases

5. Diabetes mellitus: Destruction of islets results in

pathogenesis.

6. Cataract due to aggregation of proteins.

7. Male infertility: reduce sperm motility and viability.

8. Aging process

9. Others: such as Parkinson's disease, Alzheimer’s

disease, multiple sclerosis, liver cirrhosis, muscular

dystrophy.

Severity of Oxidative Stress & Biological Consequences

Severity of

Oxidative Stress

Biological

Consequences

A. Low level & gradual Aging

B. Medium level & rapid Carcinogenesis

Mutagenesis

C. Large level & rapid Death, Stroke,

Trauma, Ionizing

irradiation

Free Radicals Anti-oxidants

(Oxidants)