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BI12.1-3 | Xenobiotic, oxidative stress and antioxidants — Self-Directed Learning

Xenobiotics and the Logic of Biotransformation

What are Xenobiotics?

Figure: Xenobiotics and the Logic of Biotransformation

Xenobiotics (Greek: xenos = foreign, bios = life) are chemicals that are foreign to the body's normal biochemistry. They include:
- Drugs: paracetamol, rifampicin, phenytoin, warfarin
- Environmental pollutants: pesticides (DDT, organophosphates), heavy metals, industrial solvents
- Dietary compounds: caffeine, ethanol, food additives, aflatoxins (from Aspergillus-contaminated groundnuts — a major problem in Indian agriculture)
- Endogenous compounds metabolised by the same enzymes: bilirubin, steroid hormones, prostaglandins

Most xenobiotics are lipophilic (fat-soluble) — they cross cell membranes easily but cannot be excreted by the kidneys (which filter water-soluble molecules). The purpose of xenobiotic metabolism is to convert lipophilic compounds into hydrophilic (water-soluble) products that can be excreted in urine or bile.

The Two-Phase System

Biotransformation occurs in two sequential phases, primarily in the liver (hepatocytes, smooth endoplasmic reticulum), though some occurs in the gut, kidneys, lungs, and skin.

Figure: The Two-Phase System

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PHASE I: Functionalisation Reactions

Phase I reactions introduce or expose a functional group (–OH, –NH₂, –SH, –COOH) on the xenobiotic. This creates a 'handle' for Phase II conjugation. The reactions include:

Figure: PHASE I: Functionalisation Reactions

1. Oxidation (most common): catalysed by the cytochrome P450 (CYP) monooxygenase system
- General reaction: RH + O₂ + NADPH + H⁺ → R-OH + H₂O + NADP⁺
- One atom of O₂ is incorporated into the substrate (hence 'monooxygenase'); the other is reduced to H₂O
- Requires: CYP450 enzyme + NADPH-cytochrome P450 reductase + phospholipid (in SER membrane) + O₂ + NADPH

1. Reduction: nitro-reduction, azo-reduction, carbonyl reduction
2. Hydrolysis: ester and amide hydrolysis (e.g., procaine → PABA + diethylaminoethanol)
3. Non-CYP oxidations: alcohol dehydrogenase (ethanol → acetaldehyde), aldehyde oxidase, monoamine oxidase (MAO), xanthine oxidase

The Cytochrome P450 Superfamily

CYP450 enzymes are a superfamily of haem-containing monooxygenases located in the smooth endoplasmic reticulum (microsomal fraction). Key facts:

Figure: The Cytochrome P450 Superfamily

• Nomenclature: CYP + family number + subfamily letter + individual enzyme number. Example: CYP3A4
• 57 human CYP genes in 18 families; only ~15 are important for drug metabolism
• CYP3A4: the single most important drug-metabolising enzyme — metabolises ~50% of all drugs (including erythromycin, cyclosporine, nifedipine, statins)
• CYP2D6: metabolises ~25% of drugs (including codeine → morphine, metoprolol, fluoxetine); shows significant genetic polymorphism — 7% of Caucasians are poor metabolisers
• CYP2C9: warfarin, phenytoin, NSAIDs
• CYP2C19: omeprazole, clopidogrel — 15–20% of Indians are poor metabolisers
• CYP1A2: caffeine, theophylline; induced by smoking
• CYP2E1: ethanol, paracetamol (produces the toxic NAPQI metabolite)

Enzyme Induction and Inhibition

CYP450 enzymes can be induced (increased synthesis) or inhibited (decreased activity), leading to clinically critical drug interactions:

Figure: Enzyme Induction and Inhibition

Inducers (↑ CYP activity)	Inhibitors (↓ CYP activity)
Rifampicin (most potent — CYP3A4)	Ketoconazole (CYP3A4)
Phenobarbital (CYP2B6, 3A4)	Erythromycin/Clarithromycin
Phenytoin (CYP3A4)	Grapefruit juice (CYP3A4)
Carbamazepine	Cimetidine (non-specific)
Chronic alcohol	Ritonavir (CYP3A4)
Smoking (CYP1A2)	Ciprofloxacin (CYP1A2)

Clinical consequence: A TB patient on rifampicin (CYP inducer) taking oral contraceptives (CYP3A4 substrate) → accelerated metabolism of OCP → contraceptive failure → unwanted pregnancy. This is one of the most commonly tested drug interactions.

Figure: Clinical consequence

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PHASE II: Conjugation Reactions

Phase II reactions attach a large, polar, water-soluble molecule to the Phase I product (or directly to the xenobiotic if it already has a suitable functional group). This dramatically increases water solubility and molecular weight, facilitating biliary or renal excretion.

Figure: PHASE II: Conjugation Reactions

Conjugation Reaction	Donor Molecule	Enzyme	Example
Glucuronidation (most common)	UDP-glucuronic acid (UDPGA)	UDP-glucuronosyltransferase (UGT)	Bilirubin, morphine, paracetamol
Sulphation	PAPS (3'-phosphoadenosine-5'-phosphosulphate)	Sulphotransferase	Steroids, paracetamol, minoxidil
Glutathione conjugation	Glutathione (GSH)	Glutathione S-transferase (GST)	NAPQI (paracetamol toxic metabolite), aflatoxin epoxide
Acetylation	Acetyl-CoA	N-acetyltransferase (NAT)	Isoniazid, sulphonamides, hydralazine
Methylation	S-adenosylmethionine (SAM)	Methyltransferases	Catecholamines (COMT), histamine
Glycine conjugation	Glycine	Acyl-CoA glycine transferase	Benzoic acid → hippuric acid

Paracetamol Toxicity — Integrating Phase I and Phase II

At therapeutic doses (≤4 g/day):
- 90% of paracetamol is conjugated by glucuronidation and sulphation (Phase II) → safe excretion
- ~5% is oxidised by CYP2E1 → NAPQI (N-acetyl-p-benzoquinone imine), a highly reactive electrophile
- NAPQI is immediately neutralised by glutathione (GSH) conjugation → mercapturic acid → excreted

Figure: Paracetamol Toxicity — Integrating Phase I and Phase II

At toxic doses (>10 g):
- Glucuronidation and sulphation pathways are saturated (substrate exhaustion)
- More paracetamol is shunted to CYP2E1 → massive NAPQI production
- Hepatic GSH stores are depleted (normally ~10 mmol/L in hepatocytes)
- Unquenched NAPQI binds covalently to hepatocyte proteins (especially mitochondrial proteins) → oxidative stress → necrosis → acute liver failure

N-acetylcysteine (NAC) is the antidote because it is a precursor of cysteine, which is the rate-limiting amino acid for glutathione synthesis. NAC replenishes GSH → NAPQI is neutralised. NAC must be given within 8–10 hours of ingestion for maximum efficacy.

Phase III: Efflux Transporters (briefly)

After Phase I and II processing, conjugated metabolites are actively pumped out of hepatocytes by ATP-binding cassette (ABC) transporters:
- P-glycoprotein (MDR1/ABCB1): pumps drugs into bile canaliculi and intestinal lumen
- MRP2 (ABCC2): exports glucuronide and glutathione conjugates into bile
- Clinical relevance: overexpression of P-gp in cancer cells → multidrug resistance (the cell pumps out chemotherapy drugs before they can act)

Reactive Oxygen Species and Oxidative Stress

Free Radicals and Reactive Oxygen Species (ROS)

Figure: Reactive Oxygen Species and Oxidative Stress

A free radical is any chemical species with one or more unpaired electrons in its outer orbital. Free radicals are highly reactive — they 'steal' electrons from neighbouring molecules, damaging them and generating chain reactions.

Key ROS and RNS (Reactive Nitrogen Species):

Species	Symbol	Source	Notes
Superoxide anion	O₂•⁻	Mitochondrial ETC (Complex I, III), NADPH oxidase, xanthine oxidase	Primary ROS; generated by one-electron reduction of O₂
Hydrogen peroxide	H₂O₂	Superoxide dismutase (SOD) acting on O₂•⁻; peroxisomes	Not a free radical itself (no unpaired electron), but a potent oxidant
Hydroxyl radical	•OH	Fenton reaction: Fe²⁺ + H₂O₂ → Fe³⁺ + •OH + OH⁻	Most reactive ROS; reacts within nanoseconds; no enzymatic defense
Peroxynitrite	ONOO⁻	Reaction of O₂•⁻ with nitric oxide (NO•)	Damages proteins by tyrosine nitration
Hypochlorous acid	HOCl	Myeloperoxidase in neutrophils (H₂O₂ + Cl⁻ → HOCl)	Bactericidal in phagolysosomes
Singlet oxygen	¹O₂	Photochemical reactions, myeloperoxidase	Important in photosensitivity reactions

Sources of ROS in the Cell

Figure: Key ROS and RNS (Reactive Nitrogen Species):

1. Mitochondrial electron transport chain: The most important physiological source. ~1–2% of electrons 'leak' from Complexes I and III and reduce O₂ to O₂•⁻ instead of completing the four-electron reduction to H₂O at Complex IV. In cells with high metabolic rates (hepatocytes, cardiomyocytes), this represents a significant daily ROS burden.

1. NADPH oxidase (NOX): Deliberate ROS production by phagocytes (neutrophils, macrophages) during the 'respiratory burst' to kill ingested bacteria. The reaction: 2 O₂ + NADPH → 2 O₂•⁻ + NADP⁺ + H⁺. Deficiency of NADPH oxidase → Chronic Granulomatous Disease (CGD) — recurrent bacterial and fungal infections because phagocytes cannot generate ROS to kill pathogens.

1. Xanthine oxidase: Converts hypoxanthine → xanthine → uric acid, generating O₂•⁻ and H₂O₂. Important in ischaemia-reperfusion injury — during ischaemia, ATP is degraded to hypoxanthine; during reperfusion, O₂ floods back and xanthine oxidase generates a burst of ROS.

1. Cytochrome P450 reactions: 'Uncoupled' CYP reactions can produce O₂•⁻ and H₂O₂ as byproducts.

1. Peroxisomes: β-oxidation of very-long-chain fatty acids produces H₂O₂ (neutralised by catalase within the peroxisome).

1. Exogenous sources: ionising radiation, UV radiation, cigarette smoke, heavy metals (iron, copper — catalyse Fenton reaction), drugs (doxorubicin, bleomycin, cisplatin), air pollutants.

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Oxidative Stress: Definition and Consequences

Oxidative stress is the imbalance between ROS production and antioxidant defense, with ROS production exceeding the body's capacity to neutralise them. The consequences are damage to all four major classes of biomolecules:

Figure: Oxidative Stress: Definition and Consequences

1. Lipid Peroxidation
- ROS (especially •OH) abstract a hydrogen atom from polyunsaturated fatty acids (PUFAs) in cell membrane phospholipids
- This initiates a chain reaction: lipid radical → peroxyl radical → lipid hydroperoxide → further radical generation
- End products: malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) — used as biomarkers of oxidative stress
- Consequences: membrane fluidity decreases, permeability increases, membrane-bound enzymes and receptors are damaged
- Clinical: lipid peroxidation of LDL cholesterol in the arterial intima is the initiating event in atherosclerosis (modified LDL is taken up by macrophage scavenger receptors → foam cells → fatty streak → atherosclerotic plaque)

2. Protein Oxidation
- Amino acid side chains (especially Cys, Met, His, Trp) are oxidised
- Protein carbonyl groups are formed — another biomarker of oxidative stress
- Consequences: enzyme inactivation, receptor dysfunction, protein aggregation
- Clinical: oxidation of lens crystallins → cross-linking → lens opacity → cataract (extremely common in India; UV exposure + smoke exposure in rural cooking environments are contributory)

3. DNA Damage
- ROS cause base modifications (8-oxo-deoxyguanosine — 8-OHdG — is the most studied), single-strand and double-strand breaks, DNA-protein cross-links
- 8-OHdG mispairs with adenine instead of cytosine → G:C → T:A transversion mutations
- If mutations occur in oncogenes or tumour suppressor genes → carcinogenesis
- Estimated 10,000–100,000 oxidative DNA lesions per cell per day — most are repaired by base excision repair (BER)

4. Carbohydrate Damage
- Glucose auto-oxidation and glycation (non-enzymatic glycosylation) generate ROS
- Advanced Glycation End-products (AGEs) accumulate in diabetes → bind RAGE receptors → NF-κB activation → inflammation
- This links oxidative stress to diabetic complications (nephropathy, retinopathy, neuropathy, vasculopathy)

Antioxidant Defense Systems

The body possesses an elaborate, multi-layered antioxidant defense system to counteract ROS. Antioxidants are classified into enzymatic and non-enzymatic categories.

Figure: Antioxidant Defense Systems

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ENZYMATIC ANTIOXIDANTS

1. Superoxide Dismutase (SOD)
- Reaction: 2 O₂•⁻ + 2H⁺ → H₂O₂ + O₂ (dismutation of superoxide)
- Three isoforms:
- SOD1 (Cu/Zn-SOD): cytoplasm — requires copper and zinc
- SOD2 (Mn-SOD): mitochondrial matrix — requires manganese (the most critical for survival; knockout is lethal in mice)
- SOD3 (EC-SOD): extracellular, Cu/Zn-dependent
- SOD converts a reactive radical (O₂•⁻) to a less reactive non-radical oxidant (H₂O₂), which is then handled by catalase or glutathione peroxidase

Figure: ENZYMATIC ANTIOXIDANTS

2. Catalase
- Reaction: 2 H₂O₂ → 2 H₂O + O₂
- Location: primarily in peroxisomes (very high concentration), also in erythrocytes
- Contains haem iron at the active site
- Very high turnover number: one molecule can decompose ~40 million H₂O₂ molecules per second
- Important when H₂O₂ concentration is high (peroxisomal β-oxidation)

3. Glutathione Peroxidase (GPx)
- Reaction: 2 GSH + H₂O₂ → GSSG + 2 H₂O (also reduces lipid hydroperoxides: LOOH → LOH)
- Selenoenzyme — requires selenium (Se) as selenocysteine at the active site
- More important than catalase at low H₂O₂ concentrations and for lipid hydroperoxide removal
- Five isoforms: GPx1 (cytoplasm), GPx2 (GI tract), GPx3 (plasma), GPx4 (membrane phospholipids — prevents lipid peroxidation), GPx5 (epididymis)

4. Glutathione Reductase
- Reaction: GSSG + NADPH + H⁺ → 2 GSH + NADP⁺
- Regenerates reduced glutathione (GSH) from oxidised glutathione (GSSG)
- Requires NADPH — supplied by the hexose monophosphate (HMP) shunt (glucose-6-phosphate dehydrogenase reaction)
- This is why G6PD deficiency causes oxidative haemolysis: erythrocytes cannot generate NADPH → cannot regenerate GSH → cannot neutralise H₂O₂ → membrane damage → haemolysis (triggered by oxidant drugs: primaquine, dapsone, sulphonamides; or fava beans — favism)

5. Thioredoxin System
- Thioredoxin (Trx) + thioredoxin reductase (TrxR, a selenoenzyme) + NADPH
- Reduces oxidised protein disulphide bonds; regulates transcription factors (NF-κB, AP-1)
- Important in cell signalling and redox regulation

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NON-ENZYMATIC ANTIOXIDANTS

1. Glutathione (GSH) — the master antioxidant
- Tripeptide: γ-glutamyl-cysteinyl-glycine
- Most abundant intracellular thiol (1–10 mM in most cells; highest in liver)
- Functions: (a) direct ROS scavenging via -SH group, (b) substrate for glutathione peroxidase, (c) Phase II conjugation of xenobiotics (via GST), (d) regeneration of other antioxidants (vitamins C and E)
- Synthesis: rate-limiting enzyme is glutamate-cysteine ligase (γ-glutamylcysteine synthetase); rate-limiting amino acid is cysteine
- GSH/GSSG ratio is a measure of cellular redox state (normal: >100:1 in cytoplasm)

Figure: NON-ENZYMATIC ANTIOXIDANTS

2. Vitamin C (Ascorbic Acid)
- Water-soluble; operates in aqueous compartments (plasma, cytoplasm)
- Directly scavenges O₂•⁻, •OH, and HOCl
- Regenerates vitamin E (tocopheroxyl radical → tocopherol)
- Required for collagen synthesis (prolyl and lysyl hydroxylase cofactor) — hence scurvy involves oxidative damage + collagen failure
- Dietary sources: amla (Indian gooseberry — one of the richest natural sources: ~600 mg/100g), citrus fruits, guava, green leafy vegetables

3. Vitamin E (α-Tocopherol)
- Fat-soluble; the most important chain-breaking antioxidant in cell membranes
- Donates a hydrogen atom to lipid peroxyl radicals → terminates the lipid peroxidation chain reaction
- Becomes the tocopheroxyl radical, which is regenerated by vitamin C
- Dietary sources: vegetable oils (sunflower, safflower), nuts, wheat germ
- Clinical: vitamin E deficiency causes spinocerebellar ataxia, peripheral neuropathy, and haemolytic anaemia (membrane fragility in RBCs)

4. β-Carotene and Carotenoids
- Quench singlet oxygen (physical quenching — absorb energy without chemical reaction)
- Operate in low oxygen tension environments (retina, skin)
- Dietary sources: carrots, papaya, mango, spinach, pumpkin

5. Uric Acid
- Powerful aqueous-phase antioxidant; scavenges •OH, O₂•⁻, and peroxynitrite
- Accounts for ~60% of plasma antioxidant capacity
- Paradox: uric acid is protective as an antioxidant, but excess causes gout

6. Other Non-Enzymatic Antioxidants
- Selenium: essential component of GPx and TrxR; deficiency → Keshan disease (cardiomyopathy in China), increased cancer risk
- Zinc and copper: essential for SOD1 and SOD3
- Manganese: essential for SOD2
- Coenzyme Q10 (Ubiquinone): electron carrier in mitochondrial ETC; also a lipid-soluble antioxidant in membranes
- Bilirubin: product of haem catabolism; antioxidant in plasma (unconjugated bilirubin scavenges peroxyl radicals)
- Albumin: binds free copper and iron → prevents Fenton reaction; also scavenges HOCl
- Polyphenols: flavonoids, catechins (green tea), curcumin (turmeric), resveratrol — Indian diet is rich in these; they chelate metal ions and scavenge free radicals

Oxidative Stress in Disease — Cancer, Diabetes, and Atherosclerosis

Oxidative Stress and Cancer (BI12.3)

Figure: Oxidative Stress in Disease — Cancer, Diabetes, and Atherosclerosis

ROS contribute to carcinogenesis at multiple stages:

1. Initiation: Oxidative DNA damage → mutations in oncogenes (e.g., RAS) and tumour suppressor genes (e.g., p53). The 8-OHdG lesion causes G:C → T:A transversion mutations. Chronic inflammation (e.g., hepatitis B/C → hepatocellular carcinoma, H. pylori → gastric cancer) generates sustained ROS via activated macrophages and neutrophils.

1. Promotion: ROS activate redox-sensitive transcription factors (NF-κB, AP-1, HIF-1α) → upregulation of genes promoting cell proliferation, angiogenesis, and survival. ROS also inhibit protein tyrosine phosphatases (by oxidising the catalytic cysteine) → sustained growth factor signalling.

1. Progression: ROS promote genomic instability, further mutations, epithelial-mesenchymal transition, and metastasis.

Specific examples:
- Aflatoxin B1 (from Aspergillus flavus on contaminated groundnuts, maize — common in India) → CYP3A4/CYP1A2 activate it to aflatoxin-8,9-epoxide → binds DNA (p53 codon 249 hotspot mutation) → hepatocellular carcinoma
- Tobacco smoke: benzo[a]pyrene → CYP1A1 → BPDE (benzo[a]pyrene diol epoxide) → DNA adducts → lung cancer
- Chronic alcohol: CYP2E1 induction → ↑ ROS + acetaldehyde (directly genotoxic) → oesophageal and hepatocellular carcinoma

Paradox of antioxidant supplementation: Large randomised trials (ATBC, CARET, SELECT) showed that high-dose antioxidant supplements (β-carotene, vitamin E, selenium) did NOT reduce cancer risk and in some cases increased it. This is because: (1) ROS also serve as tumour suppressors by triggering apoptosis in damaged cells; (2) antioxidants may protect cancer cells from ROS-induced death; (3) high-dose single antioxidants may act as pro-oxidants. The lesson: a balanced diet rich in diverse antioxidants is protective; megadose supplementation is not.

Figure: Paradox of antioxidant supplementation

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Oxidative Stress and Diabetes Mellitus Complications (BI12.3)

Hyperglycaemia generates oxidative stress through four major biochemical mechanisms (the 'unifying hypothesis' of Brownstein, 2001):

Figure: Oxidative Stress and Diabetes Mellitus Complications (BI12.3)

1. Increased polyol pathway flux: Excess glucose → sorbitol (aldose reductase, consumes NADPH) → fructose. NADPH depletion → decreased GSH regeneration → oxidative stress. Sorbitol accumulates in lens, Schwann cells, retinal pericytes → osmotic damage.

1. Increased AGE formation: Non-enzymatic glycation of proteins → Advanced Glycation End-products (AGEs). AGEs bind RAGE receptors → NF-κB → inflammatory cytokines + ROS production. AGE-modified collagen cross-links → basement membrane thickening (glomerular, retinal).

1. Increased PKC activation: Hyperglycaemia → ↑ diacylglycerol (DAG) → PKC activation → ↑ NADPH oxidase → ↑ O₂•⁻ production; also ↑ TGF-β → fibrosis.

1. Increased hexosamine pathway flux: Fructose-6-phosphate → glucosamine-6-phosphate → UDP-GlcNAc → O-GlcNAcylation of transcription factors (Sp1) → ↑ TGF-β, PAI-1.

Unifying mechanism: Brownstein proposed that all four pathways are activated by a single upstream event: mitochondrial overproduction of superoxide due to hyperglycaemia-driven excess NADH/FADH₂ input to the ETC. Excess electron flux at Complexes I and III → O₂•⁻ generation → GAPDH inhibition (via PARP activation) → diversion of glycolytic intermediates into the four pathways above.

Figure: Unifying mechanism

Clinical correlates: Diabetic nephropathy (glomerulosclerosis — the leading cause of end-stage renal disease in India), retinopathy (leading cause of blindness in working-age adults), neuropathy (peripheral sensory loss → diabetic foot ulcers → amputations), and macrovascular disease (accelerated atherosclerosis → MI, stroke).

Figure: Clinical correlates

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Oxidative Stress and Atherosclerosis (BI12.3)

The 'oxidative modification hypothesis' of atherosclerosis (Steinberg, 1989):

Figure: Oxidative Stress and Atherosclerosis (BI12.3)

1. Native LDL enters the subendothelial space (intima) of arteries
2. LDL is oxidised by ROS from endothelial cells, smooth muscle cells, and macrophages → oxidised LDL (oxLDL)
3. OxLDL is NOT recognised by the normal LDL receptor (which is subject to feedback inhibition) — instead, it is taken up by macrophage scavenger receptors (SR-A, CD36) with NO feedback inhibition → unlimited cholesterol uptake → foam cells
4. Foam cells accumulate → fatty streak → fibrous cap → atherosclerotic plaque
5. OxLDL also: (a) is chemotactic for monocytes, (b) inhibits macrophage motility (trapping them in the intima), (c) is cytotoxic to endothelial cells, (d) stimulates platelet aggregation

Clinical relevance: Statins reduce cardiovascular events partly through antioxidant effects (beyond LDL lowering). The Indian diet rich in turmeric (curcumin) and spices with antioxidant properties may partially explain the 'Indian paradox' — though this is debated, as India also has one of the highest burdens of coronary artery disease globally (attributable to metabolic syndrome, diabetes, and genetic factors like Lp(a)).

Figure: Clinical relevance