Lecture 4: Phase I Metabolism Flashcards Preview

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-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.


Phase I and Phase II Biotransformation

Reactions catalyzed by xenobiotic biotransforming enzymes are generally divided into two groups: Phase I and phase II.
1. 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.
2. 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.


Phase I and II Biotransformation

-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)


Phase I Metabolism: Cytochrome P450

-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.


Cytochrome P450 Activation

-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.


NADPH-Cytochrome P450 Reductase

-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


CYP Binding to CYP Reductase

CYP interaction with CYP reductase is mediated by:
1. Localization: CYP reductase and CYP are both membrane bound to the ER and localized together.
2. Electrostatic Interactions: CYP has a positively charged region above the heme moiety that interacts with negatively charged residues on CYP reductase.


Peroxidases (soluble)

1. Prostaglandin H synthase (PHS, COX1,2) (brain, lung, kidney, GI tract, urinary bladder)
2. Myeloperoxidase (MOx) (leukocytes)
3. 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.


Prostaglandin H synthase

PHS (COX) has two catalytic activities:
1. a cyclooxygenase (COX) that converts arachidonic acid to the cyclic endoperoxide-hydroperoxide PGG2)
2. a peroxidase (that converts the hydroperoxide to the corresponding alcohol PGH2) which can result in the oxidation of xenobiotics.
3. COX-2 inhibitors include aspirin and ibuprofin
-PHS can bioactivate carcinogens such as β-napthylamine, a bladder carcinogen


Flavin-containing Monooxygenase

-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.



-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


Epoxide Hydrolase

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.



-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


Epoxide Hydrolase Induction

-EH is inducible by 2-3 fold by:
*CYP inducers (PAH, TCCD)
-EH is inducible by 10-fold by antioxidants


Antioxidant Defenses

-Glutathione S-transferase
-Glutathione Reductase
-Quinone Reductase
-Epoxide Hydrolase



-The developments of the industrial revolution stimulated a rise in many occupational diseases.
-Percival Pott in 1775 recognized the role of soot in scrotal cancer among chimney sweeps and the problem was solved by instructing chimney sweeps to clean themselves after work.
-The causative agents were polycyclic aromatic hydrocarbons and a carcinogen culprit, benzo[a]pyrene (BaP), was isolated from coal tar in 1933.
-BaP is found in charbroiled meats, tobacco smoke, coal tar.
-BaP is a potent carcinogen upon bioactivation.



-Aflatoxins are naturally occurring mycotoxins that are produced by many species of Aspergillus, a fungus.
-They can be found on moldy peanuts, rice, corn and other crops.
-Aflatoxin B1 is the most potent liver carcinogen.