FREE RADICAL SCAVENGERS

- Dr Mangala Nischal- 2nd yr PG

- M D pharmacology

CONTENT :-• Introduction• Free Radicals & types• ROS & RNS• Sources of Free radicals• Free radical toxicity• Free radicals and diseases• Antioxidants ( In Detail)• References

Introduction • The stability of the atom is indicated by electron

pairing in the outermost orbit . • To maintain stability, each electron in the outer

orbit must be paired with another electron.

Moses Gombergthe founder of radical

chemistry (1866-1947)

• A free radical is simply an atom with one or more

unpaired electrons in its outer orbit.

• Unpaired electrons cause radicals to be highly reactive.

Free radicals

What happens when free radicals bond?

• When a weak bond is split, a free radical may be formed. • In normal circumstances, the body provides endogenous

substances (free-radical scavengers) to combine with the free radicals.

• If these scavengers aren’t available or if overproduction of free radicals radicals donate to or steal an electron from another molecule chain reaction that triggers formation of more free radicals.

• This chain reaction results in damage to the cell membrane, DNA, alter enzyme reactions, and cause damage to collagen and connective tissues.

Free Radical Nomenclature • A free radical is denoted by a superscript dot to the

oxygen or nitrogen. • e.g., HO, NO

• If a free radical is a charged species, the dot is put and then the charge

• e.g., O2-

Types of Free Radicals• Oxygen-centered radicals

• Singlet oxygen(O), superoxide(O2- ), hydroxyl radicals

• Sulfur-centered radicals• Thiyl radical (R-S•)

• Carbon-centered radicals• •CCl3, CH2

•CHOH

• Nitrogen-centered radicals• NO•, R2NO•

Reactive oxygen and nitrogen species ROS/RNSReactive oxygen species - species, which contain one or more oxygen atoms and are much more reactive than molecular oxygen

ROS/RNSFree radicals

superoxide radical

hydroperoxyl radical

hydroxyl radical

nitric oxide

hydrogen peroxide

Oxygen as a free radical

• Probably the most well-known free radical, oxygen is

the basis for development of most free radicals in the

body. Inherently, oxygen is an unstable molecule.

• During metabolism, the O2 molecule splits and energy is released. To regain stability, the free single oxygen atom (oxygen free radical) seeks out or steals electrons from other available sources. This may result in a bond with dangerous properties:

If oxygen accepts one electron, it becomes superoxide anion radical (O2

- )

If oxygen accepts two electrons, it produces peroxide. O2 2

-

• Although the superoxide radical isn’t very powerful, it can easily

donate an electron to a nearby iron atom to produce the hydroxyl

radical (OH*), one of the most potent biological free radicals.

• Hydroxyl radical (OH*) can react with almost any molecule to cause

oxidative stress and damage.

• Radicals are believed to be involved in degenerative diseases and

cancers.

• Mitochondrial electron transport chain

• Enzymatic reduction of oxygen (O2)

• Xenobiotic metabolisms (redox cycling)

• Respiratory burst (phagocytes)

Cellular sources of ROS

‘Leaking’ of electron (to oxygen) during electron transport leads to the formation of O2

- (O2 + e- O2-)

Superoxide formation from Mitochondrial Electron Transport Chain

• 0.1% to 4% of the O2 used actively by respiring mitochondria forms

O2- which is more than enough to have lethal effect on the cell unless

the free radical is quickly disposed off.

• Enzymatic reduction of oxygen

Xanthine/hypoxanthine Uric acid

• Redox cycling :

O2 O2-

XOD

[XOD = xanthine oxidase]

Respiratory Burst

NADPH oxidase complex converts

NADPH NADP+ + H+

• Electron is transferred

from NADPH to O2, resulting

in the formation of O2-

[NADPH : Reduced Nicotinamide Adenine Dinucleotide Phosphate]

Hydroxyl Radical (HO)

• Highly reactive oxygen radicals

• Formation of hydroxyl radicals in biological systems

• Ionizing radiation

• Reaction of metal ions with hydrogen peroxide (Fenton reaction)

• Formation of hydroxyl radical from ozone (O3)

• Fenton Reaction Discovered by Fenton (1894)

• “A mixture of hydrogen peroxide and an iron(II) salts causes the formation of hydroxyl radical”

Fe2+ + H2O2 Fe3+ + OH- + HO

Fe3+ + H2O2 Fe2+ + O2 - + 2H+

Cellular sources of RNS

• Nitric oxide (NO) - Nitrogen-Centered Radicals

• Endothelial derived-relaxing factor (EDRF)

• Generated from the catalysis of L-arginine by nitric oxide

synthase (NOS) enzymes

• NO + O2- ONOO- (peroxynitrite - highly toxic)

Damage(Pro-oxidants)

Defense(Antioxidants)

Oxidant-Antioxidant BalanceDamage(Pro-oxidants)

Defense(Antioxidants)

Decrease of antioxidant defense system

Oxidative damage

Oxidative Damage• Is damages caused by free radicals/reactive oxygen

species.

• Cellular damages at different levels (membrane, proteins, DNA, etc) lead to cell death, tissue injury, cellular toxicity, etc

• Reduction antioxidants

Mechanisms of cell injury mediated by ROS and RNS or Free Radical Toxicity

ROS a RNS

Modification of aa, fragmentation and

aggregation of proteins

Lipid peroxidation DNA damage

Membrane damage

Loss of membrane integrity

Damage to Ca2+ and other ion transport systems

Inability to maintain normal ion gradients

Activation/deactivation of various enzymes

Altered gene expression

Depletion of ATP

Lipids Proteins DNA

Cell injury/ Cell death

Free Radicals and Diseases

ANTIOXIDANTS

Biological Properties of Natural Antioxidants

• Natural antioxidants

• Polyphenols (phenolic, flavonoids), carotenoids, lycopene, etc

• Electron donor property

• Ability of antioxidant to donate an electron to a species (free radical) –

reducing property

• Antioxidant remains stable

Cellular Defense Mechanisms• Isolation of generation sites of reactive oxygen

species

• Inhibition of propagation phase of reactive oxygen species

• Scavenging of reactive oxygen species

• Repair of the damage caused by reactive oxygen species

• Enhancing antioxidant capability.

Types of antioxidants:

Mainly Hydrophilic and Lypophilic Antioxidant enzymes:

1. Catalase2. Glutathione peroxidase3. Glutathione reductase4. Super oxide dismutase (both Cu-Zn and Mn)

Metals binding proteins:

1. Ceruloplasmin 2. Ferritin3. Lactoferrin 4. Metallotheinein5. Transferrin 6. Hemoglobin7. Myoglobin

Common antioxidants (scavengers)

1. Bilirubin 2. Carotenoids

a. Beta-carotene b. Alpha-carotene c. Beta-cryptoxanthin d. Lutein e. Zeaxanthin f. Lycopene

3. Flavonoidsa. Quercetinb. Rutin c. Catechin

4. Uric acids 5. Thiols (R-SH) 6. Coenzyme Q10 7. Vitamin A, C, E, D.

Others antioxidants

1. Copper 2. glutathione (GSH) 3. Alpha lipoic acid 4. Manganese5. Selenium 6. Zinc

ENDOGENOUS ANTIOXIDANTS

2O2•- + 2H+ H2O2 + O2

• Superoxide Dismutase (SOD) :• Only enzyme known to react with free radical• SOD catalyses a dismutation reaction in which one O2

.- is oxidized to O2 while other is reduced to H2O2.

• SOD can afford protection against ischemic repurfusion injury, pancreatitis, MI, endotoxic shock and organ transplant.

and organ transplant.

Catalase (CAT)Function : Removes H2O2

2 H2O2 2 H2O + O2

• Prevents lipid peroxidation and protein oxidation

Glutathione Peroxidase (GPx)

• Glutathione peroxidase ( a selenium containing enzyme) scavenges toxic amts of peroxides, H2O2 and FR.

Function : Removes H2O2 & ROOH ROOH + 2 GSH ROH + H2O + GSSG

• Protects against lipid peroxidation.• Deficiency in GPX leads to oxidative hemolysis

Melatonin• Highly selective & electroactive endogenous indoleamine.

• Present in good amounts in the nervous system.

• It neutralizes nascent oxygen , removes H2O2 , NO and

inactivates peroxynitrite (ONOO.) • Stimulate brain GPX activity

Ascorbic Acid ( Vitamin C ) :

Antioxidant Function - Scavenges free radicals and reactive

oxygen molecules, which are produced during metabolic

pathways of detoxification.

• Tobacco (chewing & smoking) is rich in free radicals which

damage DNA throat & lung cancer through formation of

nitrosamine. Both vit C & E inhibit nitrosamine formation.

Alpha tocopherol (vitamin E): “Chain-breaking antioxidant”

• Prevent the peroxidation of membrane phospholipids, and

Scavenges peroxyl radical thereby inhibiting chain reaction .

• Protects against neurological damage, Atherosclerosis,

thrombotic vascular ds.

Beta Carotene :

• Ability to quench singlet oxygen, scavenge free radicals and protect

the cell membrane lipids from the harmful effects of oxidative

degradation .

Antioxidant compounds Foods containing high levels of these antioxidants

Vitamin C (ascorbic acid) Fresh Fruits and vegetables

Vitamin E (tocopherols, tocotrienols) Vegetable oils

Polyphenolic antioxidants (resveratrol, flavonoids)

Tea, coffee, soy, fruit, olive oil, chocolate, cinnamon, oregano and red wine

Carotenoids(lycopene, carotenes, lutein) Fruit, vegetables and eggs.

Natural antioxidants

Agents augmenting endogenous antioxidants:• N-acetylcysteine – is a glutathione precursor, used in treating

Paracetamol toxicity & in AIDS pt.

• Desferoxamine & Ceruloplasmin – bind to free Fe 2+ and thus inhibits

iron dependent lipid peroxidation.

EXOGENOUS ANTIOXIDANTS• Allopurinol & Oxipurinol ( antigout drugs) – Xanthine oxidase inhibitors

which inhibits superoxidation.

• Thereby protecting against repurfusion injury, MI, cerebral and

gastrointestinal reoxygenation injury.

• Selenium – enhances the antioxidant activity of vit E.

• Manganese, Zinc, copper & chromium – good sources for generating

superoxide dismutase, glutathione peroxidase and catalase enzyme.

Antioxidant from plant sources :• Garlic, Grapefruit juice, soyabean, turmeric (curcuminoids), tomato

(lycopene) & herebal preparations containing bioflavonoids.

possess good antioxidant property

• These reduce the risk of atherosclerosis, MI & various cancers.

• Spirulina – blue-green algae from shallow pond water is a good source

of SOD, B - carotene & B-complex vitamins.

References • Principles of Pharmacolgy, 2nd edition –H L Sharma, K K Sharma• Reactive Oxygen Species in Biological Systems-An Interdisciplinary Approach

(Gilbert,2002).• Free-Radical-Induced DNA Damage and Its Repair - A Chemical Perspective

(Springer, 2006)• Harpers Biochemistry 26th ed• Lehninger's Principles of Biochemistry 4th Edition - D L Nelson, Cox Lehninger - W

H Freeman 2004

Thank You

The Color Code for Antioxidants:1. Red – tomato, pink grapefruit, watermelon 2. Blue/Red/Purple/Black (BRPB) – blueberry, cherry, prune, blackberry 3. Orange/Yellow – carrot, pumpkin, orange, papaya 4. Green – broccoli, kale, spinach, pea 5. White – garlic, onion, cabbage, turnip 6. Brown/Gray – spices, nuts, seeds, endogenous sources

How to Apply the Color Code

• 1.Enzymes (Brown/Gray) • 2.Vitamins (Brown/Gray) • 3.Phenolics (BRPB) • 4.Carotenoids (Orange/Yellow, Red) • 5.Hormones (Brown/Gray• 6.Minerals (All colours) • 7.Glutathione (Brown/Gray) • 8.Lipid effectors (Orange/Yellow) • 9.Saponins, steroids and stilbenes (Green, BRPB) • 10.Sulfur-containing chemicals (Green, White)