Cytochrome P450 (CYP) and Hydroxylation

What Is Cytochrome P450?

The Core Reaction

• One atom of O₂ is incorporated into the substrate as a hydroxyl group (-OH)
• The other oxygen atom is reduced to water
• This is why CYP enzymes are called monooxygenases (only one oxygen atom goes into the substrate)

The Hydroxylase Cycle (Step-by-Step)

Class I vs. Class II P450 Systems

Nomenclature of CYP Isoforms

• CYP = cytochrome P450 root
• Number = family (≥40% amino acid identity; e.g., CYP1, CYP2, CYP3)
• Letter = subfamily (≥55% identity; e.g., CYP1A)
• Number = individual enzyme (e.g., CYP1A1)

Physiological Roles of CYP Hydroxylation

Role in Drug Metabolism (Phase 1)

• Phase 1: Hydroxylation by CYP enzymes → introduces -OH group, making the molecule more reactive
• Phase 2: Conjugation of the hydroxylated product with glucuronic acid, sulfate, or glutathione (making it water-soluble for excretion)

Induction of CYP Enzymes

• Drugs like phenobarbital cause hypertrophy of the smooth ER and a 3-4 fold increase in CYP levels within days
• This happens mainly by increased transcription but can also involve mRNA stabilization or increased translation
• Clinical importance: Inducing one CYP isoform can accelerate the metabolism of other drugs sharing that isoform - the basis of many drug-drug interactions

Key Exam Point

• Activation of vitamin D ✓
• Hydroxylation of steroid hormone precursors ✓
• Hydroxylation of xenobiotics ✓
• Hydroxylation of retinoic acid ✓
• Methylation of xenobiotics ✗ - This is a Phase 2 reaction, NOT performed by CYP enzymes

Complete Detoxification of Xenobiotics

What Are Xenobiotics?

• Drugs and medicines
• Food additives and preservatives
• Environmental pollutants (e.g., polychlorinated biphenyls/PCBs, pesticides, insecticides)
• Naturally occurring plant compounds (some beneficial as antioxidants, some toxic)
• Chemical carcinogens

Overview: Two-Phase System

Lipophilic Xenobiotic
        ↓
  [PHASE 1] - Functionalization
        ↓
  Reactive Intermediate
        ↓
  [PHASE 2] - Conjugation
        ↓
  Water-soluble Conjugate
        ↓
  Excreted in URINE or BILE

PHASE 1 - Functionalization Reactions

Main Enzymes in Phase 1

The Key CYP Reaction:

Important Note - Prodrug Activation:

• Prodrugs - inactive drugs converted to active forms (e.g., cyclophosphamide → active alkylating agent; clopidogrel → active platelet inhibitor)
• Procarcinogens - harmless compounds converted to carcinogens or mutagens (e.g., benzo[a]pyrene activated by CYP to a carcinogenic epoxide)

PHASE 2 - Conjugation Reactions

Conjugation Reactions and Their Donors

1. Glucuronidation (most common)

• Catalyzed by UDP-glucuronosyltransferases in the ER and cytosol
• UDP-glucuronic acid donates glucuronate to -OH, -NH₂, -SH, or -COOH groups
• Examples: bilirubin, aniline, benzoic acid, phenol, steroid hormones, meprobamate

2. Sulfation

• Uses PAPS (active sulfate) as donor
• Sulfotransferases conjugate alcohols, phenols, and arylamines
• Also used for steroids, glycosaminoglycans, glycolipids, glycoproteins

3. Glutathione Conjugation

• The tripeptide glutathione (GSH) is central to this pathway
• Reaction: R (electrophile) + GSH → R-S-G
• The liver has extremely high glutathione-S-transferase activity - its entire GSH pool can be depleted within minutes of heavy xenobiotic exposure
• GST enzymes also act as ligandin - they bind carcinogens, bilirubin, and steroid hormones to sequester them and prevent DNA damage
• Glutathione conjugates are exported from the liver, processed by γ-glutamyltranspeptidase (GGT) and dipeptidases, taken up by the kidney, and N-acetylated to form mercapturic acids which are excreted in urine

4. Acetylation (polymorphic)

• Reaction: X + Acetyl-CoA → Acetyl-X + CoA
• Catalyzed by acetyltransferases in hepatic cytosol
• Key clinical example: Isoniazid (INH) for tuberculosis
  • Slow acetylators - drug persists longer → greater risk of toxicity (peripheral neuropathy)
  • Fast acetylators - drug cleared quickly → may need higher doses

5. Methylation

• Uses S-adenosylmethionine (SAM) as methyl donor
• Less common; some xenobiotics only

Epoxide Hydrolase - A Protective Enzyme

Adverse Consequences When Detoxification Fails

1. Cytotoxicity (Cell Death)

• Reactive metabolites covalently bind to macromolecules (DNA, RNA, proteins)
• This triggers the DNA repair mechanism - poly(ADP-ribose polymerase) transfers ADP-ribose units to DNA-binding proteins, consuming large amounts of NAD
• Severe DNA damage depletes NAD → impairs ATP synthesis → cell death

2. Immunological / Autoimmune Effects

• A reactive xenobiotic metabolite binds to a protein as a hapten, altering its antigenicity
• The immune system generates antibodies against the modified protein
• These antibodies cross-react with the unmodified protein → potential autoimmune disease

3. Chemical Carcinogenesis

• Some chemicals are indirect carcinogens (procarcinogens) - they need CYP activation to become carcinogenic
• Example: benzo[a]pyrene (in cigarette smoke) → CYP converts it to a reactive epoxide → epoxide attacks DNA → mutation → cancer
• Epoxide hydrolase can partially counter this by detoxifying the epoxide intermediate
• Glutathione S-transferase can also sequester carcinogens, preventing them from reaching DNA

Summary Flow Chart

XENOBIOTIC (lipophilic, foreign compound)
          ↓
     LIVER (main site)
          ↓
   ┌─── PHASE 1 ──────────────────────────┐
   │  CYP450 → hydroxylation, epoxidation │
   │  FMOs → N/S/P oxidation             │
   │  Esterases → hydrolysis             │
   │  Epoxide hydrolase → dihydrodiols   │
   └──────────────────────────────────────┘
          ↓ (reactive intermediate with -OH, -NH₂, etc.)
   ┌─── PHASE 2 ──────────────────────────┐
   │  + Glucuronic acid (UGT/UDP-GlcUA)  │
   │  + Sulfate (SULT/PAPS)              │
   │  + Glutathione (GST/GSH)            │
   │  + Acetyl group (NAT/Acetyl-CoA)    │
   │  + Methyl group (MT/SAM)            │
   └──────────────────────────────────────┘
          ↓
  WATER-SOLUBLE CONJUGATE
          ↓
  Excreted in URINE (kidney) or BILE (intestine)

Key Clinical Points to Remember