Lecture 4: Phase I Metabolism Flashcards
1
Q
Biotransformation
A
- The elimination of xenobiotics often depends on their conversion to water-soluble chemicals through biotransformation, catalyzed by multiple enzymes primarily in the liver with contributions from other tissues.
- Biotransformation changes the properties of a xenobiotic usually from a lipophilic form (that favors absorption) to a hydrophilic form (favoring excretion in the urine or bile).
- The main evolutionary goal of biotransformation is to increase the rate of excretion of xenobiotics or drugs.
- Biotransformation can detoxify or bioactivate xenobiotics to more toxic forms that can cause tumorigenicity or other toxicity.
2
Q
Phase I and Phase II Biotransformation
A
Reactions catalyzed by xenobiotic biotransforming enzymes are generally divided into two groups: Phase I and phase II.
- Phase I reactions involve hydrolysis, reduction and oxidation, exposing or introducing a functional group (-OH, -NH2, -SH or –COOH) to increase reactivity and slightly increase hydrophilicity.
- Phase II reactions include glucuronidation, sulfation, acetylation, methylation, conjugation with glutathione, and conjugation with amino acids (glycine, taurine and glutamic acid) that strongly increase hydrophilicity.
3
Q
Phase I and II Biotransformation
cont
A
- With the exception of lipid storage sites and the MDR transporter system, organisms have little anatomical defense against lipid soluble toxins.
- Biotransformation is a major additional defense.
- Xenobiotic metabolism enzymes occur in highest concentration in liver, also in lung, small intestine and other sites of entry.
- Most biotransformation occurs in the endoplasmic reticulum (ER)
4
Q
Phase I Metabolism: Cytochrome P450
A
- Cytochrome P450 (CYP) enzymes are the most important in biotransformation in terms of the catalytic versatility and number of xenobiotics that it metabolizes: 400 isozymes and 36 families.
- Most CYPs are located in the liver ER (microsomes).
- CYPs are heme-containing proteins
- Microsomal and mitochondrial CYPs play key roles in biosynthesis or catabolism of steroid hormones, bile acids, fat-soluble vitamins, fatty acids and eicosanoids.
5
Q
Cytochrome P450 Activation
A
- Aliphatic hydroxylation: involves the insertion of oxygen into a C—H bond—cleavge of the C—H bond by hydrogen abstraction is the rate-limiting step
- Heteroatom oxygenation: involves abstraction of an electron from the heteroatom
- Heteroatom dealkylation: also involves abstraction of an electron from the heteroatom, but is immediately followed by abstraction of a proton (H+) from the a-carbon. Oxygen rebound leads to hydroxylation of the carbon, and rearrangement to form the corresponding aldehyde or keton with cleavage of the carbon from the heteroatom.
6
Q
NADPH-Cytochrome P450 Reductase
A
- CYP reductase transfers electrons from NADPH to CYP through redox reactions with flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN).
- CYP reductase has two domains:
- NADPH/FAD binding site
- FMN binding site
7
Q
CYP Binding to CYP Reductase
A
CYP interaction with CYP reductase is mediated by:
- Localization: CYP reductase and CYP are both membrane bound to the ER and localized together.
- Electrostatic Interactions: CYP has a positively charged region above the heme moiety that interacts with negatively charged residues on CYP reductase.
8
Q
Peroxidases (soluble)
A
- Prostaglandin H synthase (PHS, COX1,2) (brain, lung, kidney, GI tract, urinary bladder)
- Myeloperoxidase (MOx) (leukocytes)
- Lactoperoxidase (LOx) (mammary gland)
- Most oxidative biotransformations require reduced cofactors NADPH and NADH, except for peroxidases that couple the reduction of hydrogen peroxide and lipid hydroperoxides to the oxidation of other substrates called cooxidation.
9
Q
Prostaglandin H synthase
A
PHS (COX) has two catalytic activities:
- a cyclooxygenase (COX) that converts arachidonic acid to the cyclic endoperoxide-hydroperoxide PGG2)
- a peroxidase (that converts the hydroperoxide to the corresponding alcohol PGH2) which can result in the oxidation of xenobiotics.
- COX-2 inhibitors include aspirin and ibuprofin
- PHS can bioactivate carcinogens such as β-napthylamine, a bladder carcinogen
10
Q
Flavin-containing Monooxygenase
A
- FAD-containing monooxygenases (FMO) oxidize nucleophilic nitrogen, sulfur and phosphorus heteroatoms of a variety of xenobiotics.
- FMO’s are not inducible and are constitutively expressed.
- Can be inhibited by other substrates.
- Located in microsomal fraction of liver, kidney, and lung.
11
Q
Oxidases
A
- Monoamine oxidase (MAO), diamine oxidase (DAO), and polyamine oxidase (PAO) are all involved in the oxidative deamination of primary, secondary, and tertiary amines.
- MAO is located throughout the brain and is present in the liver, kidney, intestine, and blood
12
Q
Epoxide Hydrolase
A
Epoxide hydrolase (EH) catalyzes the trans-addition of water to alkene epoxides and arene oxides, which can form during Phase I (CYP/COX).
There are 5 distinct forms of EH in mammals:
1. Microsomal epoxide hydrolase (mEH)
2. Soluble epoxide hydrolase (sEH)
3. Cholesterol epoxide hydrolase
4. LTA4 hydrolase
5. Hepoxilin hydrolase
-mEH and sEH hydrolyze xenobiotic epoxides while the latter 3 hydrolases act on endogenous substrates.
-EH enzymes are found in virtually all tissues, including liver, testis, ovary, lung, kidney, skin, intestine, colon, spleen, thymus, heart and brain.
13
Q
Epoxides
A
- Epoxides are often produced during CYP oxidation and can react with DNA and protein.
- EH primarily acts as a detoxification enzyme and can rapidly convert these potentially toxic metabolites to their corresponding dihydrodiols.
- However, sometimes EH hydrolysis can lead to bioactivation
14
Q
Epoxide Hydrolase Induction
A
- EH is inducible by 2-3 fold by:
* CYP inducers (PAH, TCCD) - EH is inducible by 10-fold by antioxidants
* BHA, BHT
15
Q
Antioxidant Defenses
A
- Glutathione S-transferase
- Glutathione Reductase
- Quinone Reductase
- Epoxide Hydrolase
