Antioxidant Defense System & Detoxification

Introduction

Life in an oxygenated environment is a double-edged sword. While oxygen (O₂) is indispensable for aerobic respiration and energy production, its partial reduction inevitably leads to the formation of Reactive Oxygen Species (ROS) — unstable and highly reactive molecules capable of damaging nearly every class of biological macromolecule.

ROS have both physiological (signaling) and pathophysiological (damaging) roles. Under normal physiological conditions, a delicate balance exists between ROS generation and their neutralization by antioxidant defense systems. When this balance shifts toward excessive ROS, oxidative stress ensues — a central mechanism implicated in ageing, cardiovascular diseases, diabetes, cancer, neurodegeneration, and many other pathologies.

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Definition

Reactive Oxygen Species (ROS):
Chemically reactive molecules derived from molecular oxygen (O₂) either by one-electron or two-electron reduction steps. They may exist as free radicals (species with one or more unpaired electrons) or non-radical reactive derivatives that are potent oxidants.

Examples include superoxide anion (O₂•⁻), hydroxyl radical (•OH), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), hypochlorous acid (HOCl), and peroxynitrite (ONOO⁻).

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Types and Classification of ROS

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Biochemical Generation of ROS

ROS are constantly produced during aerobic metabolism in all cells, but their overproduction occurs during pathological conditions such as inflammation, ischemia–reperfusion, ionizing radiation exposure, or exposure to xenobiotics.

A. Mitochondrial Electron Transport Chain (ETC)

• Mitochondria are the primary source of ROS in most cells.

• During oxidative phosphorylation, electrons leak from complexes I (NADH dehydrogenase) and III (cytochrome bc₁ complex), reducing O₂ to superoxide anion (O₂•⁻).

  O2+e−→O2•−O₂ + e⁻ → O₂•⁻O2​+e−→O2​•−

• Normally, about 1–2% of the total oxygen consumed undergoes this “electron leak”.

• Superoxide dismutase (SOD) converts superoxide to hydrogen peroxide (H₂O₂), which is detoxified by catalase or glutathione peroxidase.

• However, in excessive accumulation, H₂O₂ participates in the Fenton reaction producing the hydroxyl radical (•OH)—the most destructive ROS.

  Fe2++H2O2→Fe3++•OH+OH−Fe^{2+} + H₂O₂ → Fe^{3+} + •OH + OH⁻Fe2++H2​O2​→Fe3++•OH+OH−

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B. Respiratory Burst in Phagocytes

• During infection, neutrophils, eosinophils, and macrophages undergo a respiratory burst to generate ROS as a defense mechanism.

• The enzyme NADPH oxidase catalyzes:

  O2+NADPH→O2•−+NADP++H+O₂ + NADPH → O₂•⁻ + NADP⁺ + H⁺O2​+NADPH→O2​•−+NADP++H+

• Downstream reactions:

  • Superoxide dismutation: O₂•⁻ → H₂O₂

  • Myeloperoxidase reaction: H₂O₂ + Cl⁻ → HOCl (potent bactericidal agent)

• These ROS are essential for pathogen killing but can also cause tissue injury during chronic inflammation.

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C. Microsomal and Peroxisomal Oxidations

• Peroxisomes: Generate H₂O₂ during β-oxidation of long-chain fatty acids.

• Microsomal P450 enzymes: In xenobiotic metabolism, electrons leak to O₂ forming superoxide.

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D. Non-enzymatic Sources

• Ionizing radiation (γ-rays, UV): Splits water into •OH and H• radicals.

• Environmental pollutants and cigarette smoke: Contain ROS or induce their formation.

• Transition metals (Fe²⁺, Cu⁺): Catalyze ROS formation via Fenton and Haber–Weiss reactions:

  O2•−+H2O2→O2+•OH+OH−O₂•⁻ + H₂O₂ → O₂ + •OH + OH⁻O2​•−+H2​O2​→O2​+•OH+OH−

 

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Lipid Peroxidation

Definition

Lipid peroxidation is the oxidative degradation of polyunsaturated fatty acids (PUFAs) in cell membranes by ROS. It is a self-propagating chain reaction leading to membrane rigidity, permeability changes, and eventual cell death.

Mechanism (Three Phases)

1. Initiation: ROS (especially •OH) abstracts hydrogen from PUFA → lipid radical (L•).

   LH+•OH→L•+H2OLH + •OH → L• + H₂OLH+•OH→L•+H2​O

2. Propagation: L• reacts with O₂ → lipid peroxy radical (LOO•), which reacts with another PUFA → new L• + lipid hydroperoxide (LOOH).

3. Termination: Two radicals combine to form non-radical products (e.g., alcohols, aldehydes).

End-Products

• Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) are biomarkers of oxidative stress.

 

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Other Biochemical Sources of ROS

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Mechanisms of ROS-Induced Damage

ROS can attack nearly all cellular constituents:

A. Lipid Damage

• Chain reaction leads to membrane disruption.

• Alters fluidity and ion permeability.

B. Protein Oxidation

• Oxidation of sulfhydryl (-SH) groups → disulfide crosslinks.

• Fragmentation or denaturation of enzymes.

C. DNA Damage

• Hydroxyl radicals cause strand breaks, base modifications (8-oxoguanine), and crosslinks.

• Mutagenic lesions lead to carcinogenesis and aging.

D. Carbohydrate and Nucleic Acid Damage

• ROS oxidize sugar moieties, leading to strand scission.

• Oxidative modification of ribose impairs RNA integrity.

 

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Physiological Role of ROS

1. Cell Signaling (Redox Signaling)

• ROS act as secondary messengers in many signaling pathways.

• They modulate protein phosphorylation, transcription factor activation (e.g., NF-κB, AP-1, HIF-1α), and gene expression.

• Low levels of ROS help regulate normal cell proliferation and differentiation.

2. Immune Defense (Respiratory Burst)

• Phagocytic cells (neutrophils, macrophages) generate ROS during the “respiratory burst.”

• These ROS (superoxide, hydrogen peroxide, hypochlorous acid) help kill invading pathogens (bacteria, fungi, viruses).

3. Regulation of Vascular Tone

• Endothelial cells produce ROS (especially H₂O₂) that help modulate vascular smooth muscle relaxation.

• ROS interact with nitric oxide (NO) to maintain blood pressure homeostasis and vascular signaling.

4. Cellular Growth and Differentiation

• Moderate levels of ROS promote cell cycle progression, differentiation of stem cells, and tissue repair processes.

• Example: ROS-mediated activation of MAPK/ERK pathways promotes cell proliferation.

5. Hormonal and Metabolic Regulation

• ROS participate in insulin signaling and glucose metabolism.

• They influence thyroid hormone synthesis, steroidogenesis, and mitochondrial bioenergetics.

6. Apoptosis and Autophagy

• ROS act as signaling molecules to trigger programmed cell death (apoptosis) when cells are damaged or aged.

• Controlled ROS generation ensures removal of dysfunctional or cancerous cells.

7. Oxygen Sensing and Hypoxia Response

• ROS from mitochondria act as oxygen sensors.

• They regulate hypoxia-inducible factors (HIFs), promoting adaptive responses during low oxygen conditions.

8. Defense Against Tumor Formation (at Physiological Levels)

• Mild oxidative stress can activate tumor-suppressor pathways (e.g., p53), limiting proliferation of abnormal cells.

9. Modulation of Enzyme Activity

• ROS reversibly oxidize cysteine residues in enzymes and signaling proteins, thereby regulating their activity (redox modulation).

10. Maintenance of Cellular Homeostasis

• At physiological levels, ROS help maintain the balance between oxidation and reduction inside the cell, crucial for metabolic stability.

 

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Antioxidant Defense Systems

A. Enzymatic Antioxidants

1. Superoxide Dismutase (SOD)

   • Catalyzes:

     2O2•−+2H+→H2O2+O2​

   • Isoforms:

     • Cu/Zn-SOD – cytosolic

     • Mn-SOD – mitochondrial

     • EC-SOD – extracellular

2. Catalase

   • Located in peroxisomes.

   • Converts hydrogen peroxide → water + oxygen:

     2H2O2→2H2O+O2​

3. Glutathione Peroxidase (GPx)

   • Selenium-dependent enzyme.

   • Reduces H₂O₂ and lipid hydroperoxides:

     2GSH+H2O2→GSSG+2H2O

4. Glutathione Reductase

   • Regenerates reduced glutathione (GSH) from oxidized glutathione (GSSG) using NADPH.

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B. Non-Enzymatic Antioxidants

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C. Synergistic Actions

• Vitamin E + Vitamin C: Vitamin C regenerates oxidized α-tocopherol.

• Selenium + Vitamin E: Combined activity enhances glutathione peroxidase and membrane stability.

• GSH + Lipoic acid: Lipoic acid helps recycle oxidized glutathione and other antioxidants.

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Diseases Linked to ROS and Oxidative Stress

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Oxidative Stress Biomarkers

• Malondialdehyde (MDA)

• 4-Hydroxynonenal (4-HNE)

• Protein carbonyls

• 8-Hydroxy-2′-deoxyguanosine (8-OHdG)

• Total antioxidant capacity (TAC)

• Reduced/oxidized glutathione ratio (GSH/GSSG)

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Part II – Detoxification of Xenobiotics

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Humans are continually exposed to xenobiotics—foreign compounds such as drugs, pollutants, pesticides, food additives, and endogenous metabolic by-products. These compounds may be toxic unless biotransformed into more hydrophilic, excretable metabolites.

The liver is the principal organ for detoxification, though the kidney, lungs, intestines, and skin also contribute.

Phases of Xenobiotic Metabolism

Phase I – Functionalization Reactions

Purpose: Introduce or expose polar functional groups (–OH, –NH₂, –COOH).

1. Oxidation Reactions

   • Mediated by cytochrome P450 monooxygenases (CYPs) in the smooth endoplasmic reticulum.

     RH+O2+NADPH+H+→ROH+H2O+NADP+RH + O₂ + NADPH + H⁺ → ROH + H₂O + NADP⁺RH+O2​+NADPH+H+→ROH+H2​O+NADP+

   • Examples:

     • Methanol → Formaldehyde → Formic acid

     • Ethanol → Acetaldehyde → Acetic acid

     • Aniline → p-Aminophenol

2. Reduction Reactions

   • Nitros, carbonyls, and azo compounds reduced to less reactive species.

     • Nitrobenzene → Aniline

     • Aldehydes/Ketones → Alcohols

3. Hydrolysis Reactions

   • Catalyzed by esterases, amidases, and peptidases.

     • Aspirin → Salicylic acid + Acetic acid

     • Procaine → PABA + diethylaminoethanol

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Phase II – Conjugation Reactions

Purpose: Couple xenobiotic or Phase I metabolite with endogenous hydrophilic compounds → highly polar, water-soluble conjugates.

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Cytochrome P450 System

Characteristics

• Heme-containing monooxygenase family (> 50 isoenzymes).

• Located in smooth endoplasmic reticulum and mitochondria.

• Require NADPH, O₂, and flavoprotein (cytochrome P450 reductase).

• Inducible by drugs (e.g., phenobarbital, rifampicin) and inhibited by others (cimetidine, erythromycin).

Reaction:

RH + O₂ + NADPH + H⁺     →   ROH + H₂O + NADP⁺

Clinical Relevance

• Determines drug half-life, drug–drug interactions, and metabolic polymorphisms.

• Overactivity: ↑ Toxic intermediates (e.g., acetaminophen → NAPQI).

• Underactivity: Drug accumulation and toxicity.

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Detoxification Examples

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Biological Significance of Reactive Oxygen Species

Although ROS are often considered harmful, they are not merely toxic by-products. At physiological concentrations, they play indispensable roles in normal cellular signaling, host defense, and gene regulation — a concept termed “redox biology”.

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A. Physiological and Regulatory Roles

1. Cell Signaling and Redox Modulation

   • ROS act as second messengers in several intracellular signaling cascades.

   • Hydrogen peroxide (H₂O₂), being membrane-permeable, modulates protein phosphorylation by reversibly oxidizing cysteine residues of phosphatases (e.g., PTP1B), thereby influencing kinase signaling (MAPK, PI3K-Akt).

   • ROS regulate transcription factors like:

     • NF-κB (Nuclear Factor kappa B): Governs genes for cytokines, inflammation, apoptosis.

     • HIF-1α (Hypoxia-Inducible Factor): Activated under hypoxia, induces erythropoietin and VEGF.

     • Nrf2 (Nuclear Factor E2-related Factor 2): Master regulator of antioxidant defense gene transcription.

2. Host Defense Mechanism

   • ROS are vital in phagocytic bactericidal activity.

   • NADPH oxidase in neutrophils generates superoxide and H₂O₂, which, via myeloperoxidase, forms hypochlorous acid (HOCl) — a potent antimicrobial oxidant.

   • This process forms the biochemical basis of the respiratory burst.

3. Apoptosis and Cellular Differentiation

   • Moderate ROS levels trigger mitochondrial permeability transition, release of cytochrome c, and activation of caspase cascades—a controlled mechanism of cell death.

   • ROS modulate differentiation of myoblasts, adipocytes, and osteoclasts by altering redox-sensitive transcription factors.

4. Vasoregulation

   • Endothelial nitric oxide (NO•) reacts with superoxide to form peroxynitrite (ONOO⁻), influencing vascular tone.

   • Controlled ROS generation maintains endothelial homeostasis, angiogenesis, and smooth muscle relaxation.

5. Defense Against Xenobiotics

   • Controlled ROS participate in Phase I detoxification via cytochrome P450 enzymes.

   • This redox-based system allows oxygen insertion into lipophilic substrates for subsequent conjugation.

6. Immunity and Hormonal Signaling

   • ROS enhance antigen presentation, modulate T-cell receptor (TCR) signaling, and regulate immune tolerance.

   • They also modulate insulin signaling (via oxidation of PTEN) and thyroid hormone synthesis (via H₂O₂-dependent iodination).

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B. Beneficial Aspects of ROS (“Redox Signaling”)

• ROS at physiological levels maintain cellular adaptation (hormesis).

• Exercise-induced mild oxidative stress enhances mitochondrial biogenesis and antioxidant enzyme synthesis.

• Thus, complete ROS elimination may disrupt beneficial redox signaling — emphasizing the importance of balance, not eradication.

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Biological Significance of Detoxification

Detoxification is not merely defensive; it is an adaptive and regulatory metabolic process with wide biological implications.

A. Protection Against Environmental and Dietary Toxins

• Prevents accumulation of harmful xenobiotics from food, air, water, and drugs.

• Converts lipophilic compounds into hydrophilic metabolites for renal or biliary excretion.

B. Maintenance of Metabolic Homeostasis

• Phase I and II detox pathways maintain chemical homeostasis by balancing oxidative and conjugative metabolism.

• These pathways are linked to energy metabolism, NADPH generation, and glutathione turnover.

C. Hormone and Endobiotic Metabolism

• Endogenous compounds like steroids, bilirubin, and fatty acids undergo the same P450-mediated oxidation and conjugation.

• Thus, detoxification influences endocrine function, lipid metabolism, and bile acid turnover.

D. Regulation of Cellular Redox State

• Cytochrome P450 and flavin-containing monooxygenases (FMOs) continuously consume NADPH, thereby linking detoxification to oxidative balance.

• Excessive ROS from P450 reactions are controlled by antioxidant systems, integrating both processes.

E. Pharmacological and Nutritional Relevance

• Determines drug bioavailability, half-life, and toxicity.

• Explains interindividual variations in drug response due to genetic polymorphisms (e.g., CYP2D6, CYP3A4).

• Nutrients like selenium, flavonoids, and cruciferous vegetable isothiocyanates enhance detoxification enzymes.

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Clinical Correlations of ROS and Detoxification

ROS and detoxification pathways play dual roles — both protective and pathogenic — depending on context, tissue specificity, and enzyme regulation.

A. Cardiovascular System

• Atherosclerosis: Oxidized LDL (ox-LDL) induces endothelial injury, foam cell formation, and plaque development.

• Ischemia–Reperfusion Injury: Burst of ROS upon reoxygenation damages myocardium via lipid peroxidation and Ca²⁺ overload.

• Hypertension: ROS inactivate nitric oxide, impair vasodilation, and promote vascular remodeling.

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B. Central Nervous System

• Neurodegenerative Diseases:

  • Alzheimer’s disease: ROS cause β-amyloid aggregation and mitochondrial DNA damage.

  • Parkinson’s disease: Dopamine auto-oxidation produces quinones and peroxides that kill substantia nigra neurons.

  • Amyotrophic lateral sclerosis (ALS): Mutations in SOD1 impair superoxide dismutation.

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C. Hepatic System

• Drug-Induced Liver Injury (DILI): Acetaminophen overdosage leads to NAPQI accumulation, depleting glutathione and causing necrosis.

• Alcoholic Liver Disease: Ethanol metabolism generates acetaldehyde and ROS through CYP2E1 and MEOS (microsomal ethanol oxidizing system).

• Non-Alcoholic Fatty Liver Disease (NAFLD): Lipid peroxidation induces inflammation and fibrosis.

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D. Endocrine and Metabolic Disorders

• Diabetes Mellitus:

  • Chronic hyperglycemia increases mitochondrial ROS.

  • Damages pancreatic β-cells and induces insulin resistance.

  • Glycation end-products (AGEs) amplify oxidative stress.

• Obesity and Metabolic Syndrome:

  • Excess adipose tissue releases ROS and inflammatory cytokines.

  • Impairs endothelial nitric oxide production and lipid metabolism.

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E. Renal and Pulmonary Systems

• Renal Ischemia: ROS-induced tubular necrosis during reperfusion.

• Cigarette Smoke Exposure: Contains free radicals; damages alveoli, leading to emphysema and COPD.

• ARDS (Acute Respiratory Distress Syndrome): Neutrophil-derived ROS increase vascular permeability and pulmonary edema.

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F. Reproductive Health

• Male Infertility: ROS damage sperm DNA and mitochondrial membrane, reducing motility.

• Female Reproductive Aging: ROS impair oocyte quality and hormonal balance.

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G. Cancer and Mutagenesis

• DNA Oxidation: ROS generate base modifications (8-oxo-dG), strand breaks, and chromosomal instability.

• Oncogene Activation: Redox imbalance activates MAPK, NF-κB, and AP-1 pathways promoting proliferation.

• Tumor Suppressor Inactivation: Oxidation of p53 and PTEN impairs apoptosis and DNA repair.

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H. Aging and Degenerative Processes

• The Free Radical Theory of Aging attributes tissue aging to cumulative oxidative damage.

• ROS damage mitochondrial DNA, impair respiratory enzymes, and promote lipofuscin accumulation in senescent cells.

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I. Inflammatory and Autoimmune Disorders

• Chronic inflammation sustains ROS generation by macrophages.

• ROS oxidize membrane lipids and extracellular matrix, perpetuating inflammatory cycles.

• Seen in rheumatoid arthritis, ulcerative colitis, and glomerulonephritis.

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Detoxification-Linked Disorders

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K. Genetic and Nutritional Modulation

• Polymorphisms in P450 genes (CYP2C9, CYP2D6, CYP3A4) affect drug efficacy and toxicity.

• Selenium and Vitamin E deficiency weakens GPx defense, enhancing lipid peroxidation.

• Flavonoids, cruciferous vegetables (e.g., broccoli, cabbage) induce Phase II detoxification enzymes (via Nrf2).