FREE RADICALS AND ANTIOXIDANTS

Complete 10-Mark Answer (M.Pharm Level)

1. DEFINITION OF FREE RADICALS

Key Point: The term "free radical" is broader than ROS - it includes both oxygen-derived and nitrogen-derived reactive species.

2. CHARACTERISTICS OF FREE RADICALS

3. CLASSIFICATION OF FREE RADICALS

A. Reactive Oxygen Species (ROS)

B. Reactive Nitrogen Species (RNS)

Remember: NO itself has beneficial roles (vasodilation, signaling) but becomes harmful when it reacts with O₂•⁻ to form the very destructive peroxynitrite (ONOO⁻).

4. SOURCES / GENERATION OF FREE RADICALS

A. Endogenous Sources

• During normal aerobic respiration, O₂ is reduced to H₂O via 4-electron transfer
• Around 1-2% of electrons "leak" prematurely and do single-electron reduction of O₂
• This incomplete reduction produces superoxide (O₂•⁻) at Complex I and III

• Activated neutrophils and macrophages produce a rapid "oxidative burst"
• NADPH oxidase transfers electrons from NADPH to O₂ → O₂•⁻
• Purpose: to kill invading microorganisms (antimicrobial defense)

• Converts hypoxanthine → xanthine → uric acid during purine catabolism
• Produces O₂•⁻ and H₂O₂ as byproducts
• Important in ischemia-reperfusion injury

• Oxidative enzymes in peroxisomes generate H₂O₂ during fatty acid oxidation
• Normally degraded by catalase present within peroxisomes

• Drug metabolism in the ER generates ROS as byproducts
• Example: CCl₄ → CCl₃• (trichloromethyl radical) - causes liver injury

• Produces NO• in endothelial cells, neurons, macrophages
• NO• + O₂•⁻ → ONOO⁻ (peroxynitrite - highly destructive RNS)

• Fenton Reaction: H₂O₂ + Fe²⁺ → Fe³⁺ + •OH + OH⁻
• Iron and copper catalyze the generation of the most reactive hydroxyl radical (•OH)

B. Exogenous Sources

• Ionizing radiation (X-rays, gamma rays) - hydrolyzes water → •OH + H•
• UV radiation - generates singlet oxygen and •OH
• Cigarette smoke - contains >1000 free radicals per puff, >4000 oxidant compounds
• Drugs and toxins - CCl₄, paraquat, adriamycin
• Air pollutants - ozone (O₃), nitrogen oxides
• Heavy metals (lead, mercury, cadmium)
• Reperfusion injury - after restoration of blood flow to ischemic tissue

5. FLOWCHART OF OXIDATIVE STRESS

STIMULI: Radiation, Toxins, Inflammation, Ischemia-Reperfusion, Metabolism
                              ↓
           EXCESS PRODUCTION OF ROS / RNS
      (O₂•⁻, H₂O₂, •OH, ONOO⁻)
                              ↓
         Overwhelms Antioxidant Defense Systems
      (SOD, Catalase, GPx, Vitamin C, Vitamin E)
                              ↓
           ┌─────────OXIDATIVE STRESS─────────┐
           ↓                ↓                 ↓
    Lipid Peroxidation  Protein Damage    DNA Damage
    (membrane damage)  (enzyme inhibition) (strand breaks,
                       (protein cross-      mutations,
                       linking)             adducts)
           ↓                ↓                 ↓
    Cell Membrane        Enzyme/              Apoptosis /
    Destruction       Structural protein      Necrosis /
                        dysfunction           Mutagenesis
                              ↓
              PATHOLOGICAL OUTCOMES:
     Cancer | Diabetes | CVD | Neurodegeneration | Aging

6. HARMFUL EFFECTS OF FREE RADICALS

A. Lipid Peroxidation

• •OH attacks polyunsaturated fatty acids (PUFA) in cell membranes
• Generates malondialdehyde (MDA) and lipid peroxides as end products
• Chain reaction: One radical damages hundreds of lipid molecules
• Result: Cell membrane disruption → increased permeability → cell lysis

B. Protein Damage

• Free radicals oxidize amino acid side chains (especially cysteine, methionine, tyrosine)
• Cause covalent protein-protein cross-links (disulfide bonds)
• Damage active sites of enzymes → enzyme inactivation
• Cause protein unfolding → targets for proteasomal degradation
• Disrupts structural proteins → cytoskeletal damage

C. DNA Damage

• Cause single-strand and double-strand DNA breaks
• Cross-link DNA strands (block transcription and replication)
• Form DNA adducts (base modifications like 8-OHdG)
• Lead to mutations → cancer initiation
• Contribute to cellular aging (accumulation of damaged DNA over time)

D. Mitochondrial Damage

• Mitochondrial membranes are rich in PUFA → especially vulnerable to lipid peroxidation
• Damage to mitochondrial DNA (mtDNA) - no histones for protection
• Opening of mitochondrial permeability transition pore → ATP depletion
• Release of cytochrome c → triggers apoptosis

E. Carbohydrate Damage

• Oxidation of glucose and other sugars
• Glycation of proteins (contributes to AGE formation in diabetes)
• Damage to hyaluronic acid in joints → inflammatory joint disease

7. ROLE OF FREE RADICALS IN DISEASES

Free Radicals in Cancer - Detail

• ROS cause DNA base modifications (8-OHdG is a biomarker of oxidative DNA damage)
• These mutations may activate proto-oncogenes (e.g., Ras) or inactivate tumor suppressor genes (e.g., p53)
• ROS activate NF-κB signaling → promotes cell survival, proliferation, and metastasis
• ROS promote angiogenesis via HIF-1α activation
• Paradoxically, very high ROS levels in cancer cells can also trigger apoptosis (basis of some chemotherapy)

Free Radicals in Diabetes Mellitus - Detail

• Glucotoxicity mechanism: High blood glucose undergoes auto-oxidation → generates O₂•⁻ and H₂O₂
• Pancreatic β-cells are especially vulnerable because they have low antioxidant enzyme levels (low catalase, low GPx)
• ROS cause β-cell mitochondrial dysfunction → impaired ATP generation → reduced insulin secretion
• In Type 2 DM: ROS activate stress kinases (JNK, IKK) → impair insulin signaling (insulin resistance)
• Vascular complications: ROS oxidize LDL → atherosclerosis; damage endothelial cells → diabetic nephropathy, retinopathy, neuropathy

8. DEFINITION OF ANTIOXIDANTS

"An antioxidant is a substance produced in sufficient quantity that neutralizes the lone electron of free radicals." - Tietz Textbook of Laboratory Medicine

9. CLASSIFICATION OF ANTIOXIDANTS

A. Based on Mechanism

• Metal chelators: Transferrin, Ceruloplasmin, Ferritin (bind Fe, Cu to prevent Fenton reaction)
• Albumin (binds heme groups and copper)
• Lactoferrin (binds iron in inflammatory exudates)

• Vitamin E (α-tocopherol)
• Vitamin C (ascorbic acid)
• Beta-carotene, flavonoids
• Glutathione (GSH)

• DNA repair enzymes (e.g., 8-OHdG glycosylase)
• Proteasomes (remove damaged proteins)
• Phospholipases (remove oxidized fatty acids)

B. Based on Enzyme Activity

I. Enzymatic Antioxidants

II. Non-Enzymatic Antioxidants

C. Based on Source

10. MECHANISM OF ACTION OF ANTIOXIDANTS

A. SOD - First Line of Defense

O₂•⁻  + O₂•⁻  + 2H⁺  →  H₂O₂ + O₂
         (SOD catalyzes this dismutation)

• SOD converts the very reactive superoxide to the less reactive H₂O₂
• Cu/Zn-SOD works in cytoplasm; Mn-SOD works in mitochondria
• Extracellular SOD (EC-SOD) protects blood vessel walls

B. Catalase and Glutathione Peroxidase - Second Line

Catalase:     2H₂O₂ → 2H₂O + O₂     (in peroxisomes)
GPx:          H₂O₂ + 2GSH → 2H₂O + GSSG   (in cytosol + mitochondria)
              ROOH + 2GSH → ROH + H₂O + GSSG  (lipid hydroperoxides)

• Glutathione Reductase regenerates GSH from GSSG using NADPH (from pentose phosphate pathway)

C. Vitamin E (Fat-Soluble Chain Breaker)

• Located within cell membranes (lipid bilayer)
• Donates a hydrogen atom (H•) to a lipid peroxyl radical (LOO•)
• Converts LOO• to LOOH (stable lipid hydroperoxide)
• Vitamin E radical (Toc•) is relatively stable and is regenerated by Vitamin C

LOO• + Vit-E-OH  →  LOOH + Vit-E-O•
Vit-E-O• + Vit-C  →  Vit-E-OH + Vit-C•    (Vit C regenerates Vit E)
Vit-C• + GSH → Vit-C + GS•                (GSH regenerates Vit C)

D. Vitamin C (Water-Soluble Scavenger)

• Directly scavenges O₂•⁻, •OH, HOCl in aqueous phase
• Regenerates Vitamin E (most important synergistic action)
• High concentrations in plasma (~60 µmol/L) provide continuous protection

E. Metal Chelation (Preventive Mechanism)

• Transferrin, Ferritin, Ceruloplasmin, Albumin, Lactoferrin
• Bind free Fe²⁺/Fe³⁺ and Cu²⁺ tightly
• Prevent these metals from catalyzing Fenton/Haber-Weiss reactions
• No free metal → no •OH generation

F. Glutathione System (Most Versatile)

• GSH acts as direct scavenger of •OH, HOCl, and singlet O₂
• Acts as co-substrate for GPx to neutralize H₂O₂ and lipid peroxides
• Conjugates with electrophilic compounds via glutathione-S-transferase (GST)
• GSSG is recycled back to GSH by glutathione reductase (NADPH-dependent)

11. THERAPEUTIC ROLE OF ANTIOXIDANTS

A. In Cancer

• Prevention: Dietary antioxidants (Vitamin C, E, beta-carotene, selenium) reduce oxidative DNA damage → reduce cancer risk
• Adjunct to chemotherapy: N-acetylcysteine (NAC) protects normal cells from chemotherapy-induced ROS
• Caution: High-dose antioxidants may protect cancer cells too - controversial in active treatment
• Edaravone: Used as a free radical scavenger in some oncologic settings

B. In Cardiovascular Disease

• Vitamin E: Reduces LDL oxidation, reduces foam cell formation → anti-atherosclerotic
• Vitamin C: Improves endothelial function, reduces vascular oxidative stress
• Coenzyme Q10: Reduces oxidative stress in heart failure; used as adjunct in myocardial protection
• Resveratrol, flavonoids: Activate Nrf2 pathway → upregulate endogenous antioxidant enzymes

C. In Diabetes Mellitus

• Alpha-lipoic acid (ALA): Both water and fat soluble; regenerates Vitamin C and E; improves insulin sensitivity; used clinically for diabetic neuropathy
• Vitamin E: Reduces oxidative stress in β-cells; may improve glycemic control
• NAC: Protects β-cells from oxidative damage

D. In Neurodegeneration

• Vitamin E: Slows cognitive decline in Alzheimer's disease (modest effect)
• Edaravone (Radicava): FDA-approved free radical scavenger for ALS (Amyotrophic Lateral Sclerosis)
• Melatonin: Neuroprotective via multiple antioxidant mechanisms in Parkinson's disease

E. In Reperfusion Injury

• Pre-treatment with SOD mimetics or NAC reduces ischemia-reperfusion injury
• Allopurinol (xanthine oxidase inhibitor) reduces ROS generation on reperfusion

F. In Liver Disease

• NAC (N-acetylcysteine): Standard treatment for paracetamol (acetaminophen) overdose - replenishes GSH stores depleted by toxic metabolite NAPQI
• Silymarin (milk thistle): Scavenges free radicals, stabilizes hepatocyte membranes in liver disease

G. Key Clinical Antioxidant Drugs

12. CONCLUSION

Quick Revision Mnemonics

• Superoxide (O₂•⁻)
• Hydrogen peroxide (H₂O₂)
• Hydroxyl radical (•OH)
• Ozone, singlet oxygen

• SOD (Superoxide Dismutase)
• CAT (Catalase)
• GPx (Glutathione Peroxidase)

• Carotene
• Ascorbic acid (Vit C)
• Glutathione
• E-vitamin (tocopherol)
• Uric acid
• Flavonoids

What's Inside (8 Slides)

FREE RADICALS & ANTIOXIDANTS — TOPPER SUMMARY

Read this 15 mins before exam. Everything you need, nothing you don't.

THE ONE-LINE CONCEPT

Free radical = unstable molecule with unpaired electron → attacks cells → disease. Antioxidant = neutralizes it → protection.

RAPID-FIRE DEFINITIONS

CLASSIFICATION (The 6 ROS + 4 RNS)

ROS — Remember: "Super Hot OH Once Sing"

RNS — Remember: "NO PANDA"

GENERATION — 5 KEY SOURCES

1. MITOCHONDRIA (ETC)  →  1-2% electron leak  →  O₂•⁻   ← MOST IMPORTANT
2. NADPH OXIDASE       →  Phagocytes (PMNs)  →  O₂•⁻   ← Antimicrobial burst
3. XANTHINE OXIDASE    →  Ischemia-reperfusion →  O₂•⁻   ← Organ injury
4. FENTON REACTION     →  Fe²⁺ + H₂O₂  →  •OH          ← Most reactive radical
5. NOS (Nitric Oxide Synthase) →  NO•  +  O₂•⁻  →  ONOO⁻  ← RNS

Fenton Reaction is HIGH YIELD: Fe²⁺ + H₂O₂ → Fe³⁺ + •OH + OH⁻

OXIDATIVE STRESS FLOWCHART

Triggers: Radiation / Toxins / Inflammation / Metabolism / Ischemia
                          ↓
              EXCESS ROS / RNS Production
                          ↓
         Overwhelms Antioxidant Defense
                          ↓
         ┌────OXIDATIVE STRESS────┐
         ↓           ↓           ↓
  Lipid Peroxidation  Protein  DNA Damage
  (membrane lysis)   Damage   (mutations)
         ↓
  CANCER | DIABETES | CVD | NEURODEGENERATION | AGING

HARMFUL EFFECTS — 3 TARGETS (LCD)

ANTIOXIDANT ENZYMES — THE "SOD-CAT-GPx-GR" CASCADE

O₂•⁻  ──[SOD]──→  H₂O₂  ──[CATALASE]──→  H₂O + O₂
                     │
                   [GPx]  ──(+GSH)──→  H₂O + GSSG
                                              │
                                           [GR + NADPH]
                                              │
                                           ↙ GSH recycled ↺

NON-ENZYMATIC ANTIOXIDANTS — MNEMONIC: "CAGE UF"

THE REGENERATION CHAIN (VERY HIGH YIELD)

LOO• (Lipid radical)
   ↓ neutralized by
VITAMIN E  →  Vit-E-O• (stable)
   ↓ regenerated by
VITAMIN C  →  Asc•  (semi-stable)
   ↓ regenerated by
GLUTATHIONE (GSH)  →  GSSG
   ↓ regenerated by
GLUTATHIONE REDUCTASE + NADPH  →  GSH again ↺
   ↑
NADPH comes from Pentose Phosphate Pathway (G6PD enzyme)

Exam line: "Vitamin E, C, and GSH form a synergistic regeneration cascade — each antioxidant recycles the one before it."

DISEASES TABLE — ROLE IN DISEASE

CLINICAL ANTIOXIDANT DRUGS — MUST KNOW

LAST-MINUTE HIGH-YIELD FACTS

1. •OH = most reactive and most dangerous ROS (no enzyme inactivates it directly)
2. SOD = first line enzyme defense — converts O₂•⁻ before it can form •OH
3. GPx needs Selenium — selenium deficiency = impaired antioxidant defense
4. Catalase is only in peroxisomes — not mitochondria
5. β-cells are extra vulnerable — have LOW catalase and GPx levels
6. Fenton reaction generates •OH from H₂O₂ using Fe²⁺ — most important source of •OH
7. ONOO⁻ (peroxynitrite) = NO• + O₂•⁻ — no enzymatic inactivation; PRDx handles it
8. Nrf2 = master transcription factor for antioxidant gene expression
9. MDA = end product of lipid peroxidation (clinical biomarker)
10. 8-OHdG = oxidative DNA damage biomarker
11. NAC within 8-10 hours of paracetamol overdose is life-saving
12. LDL oxidation by ROS = first step in atherosclerosis

TOPPER EXAM TRICK — 3 LAYERS OF DEFENSE

LAYER 1 — PREVENTION:     Metal chelators (Transferrin, Ceruloplasmin, Albumin)
                           → Block Fenton reaction → No •OH formed

LAYER 2 — INTERCEPTION:   SOD, Catalase, GPx, GR
                           → Enzymatically destroy ROS as they form

LAYER 3 — REPAIR:         DNA repair enzymes, Proteasome, Phospholipase
                           → Fix damage that already happened

ANSWER STRUCTURE FOR EXAM (follow this order)

Time-saving tip: Draw the SOD→CAT/GPx→GR cascade as a diagram in your answer. It saves 150 words and earns more marks than pure text.