Xenobiotic metabolism Flashcards by Natasja Jacobsen

What is the definition of biotransformation?

The process where the body attempts to convert lipophilic substances into more polar metabolites that can be excreted quicker

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What is the primary result of biotransformation?

1.The parent molecule is transformed into a more polar metabolite, often by the addition of ionizable functional groups
2. Molecular weight and size are often increased
3. The excretion is facilitated, and hence elimination of the compound from the tissue

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What are the consequences of metabolism?

The biological half-life is decreased
The duration of exposure is reduced
Accumulation of the compound in the body is avoided
The duration of the biological activity may be affected.
Detoxification –> Less toxic/inactivation
Toxification –> Can sometimes lead to more toxic compounds or another toxic effect

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What characterize enzymes involved in metabolism?

Enzymes involved in metabolism are flexible and their substrate specificity is generally broad

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What is the location of enzymes in biotransformation?

Many are found in the smooth endoplasmic reticulum (SER)
Some in the cytosol
Few in other organelles e.g. mitochondria

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What are the main types of phase 1 reactions?

1) Oxidation
2) Reduction
3) Hydrolysis

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Which types of phase 2 reactions are there?

1) Glucuronidation
2) Sulfation
3) Glutathione conjugation
4) Conjugation with amino acids
5) Methylation
6) Acetylation

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What is the purpose of phase 1 reactions

Alteration of the original molecule by exposing or introducing a functional group (-OH, -NH2, -SH, -COOH)

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What is the purpose of phase 2 reactions

Conjugation of a polar group to the functional group –> Increases hydrophilicity (except for methylation and acetylation) –> facilitates excretion

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Which enzymes are usually included in Phase 1 drug metabolism?

Cytochrome P450s (CYP450 or CYPs)
Alcohol dehydrogenase (ADH)
Aldehyd dehydrogenase (ALDH)
Esterases/amidases (hydrolases)
Amine oxidase (MAO, FMO)
Xanthine oxidase (XO) – a pyrine
Alkylhydrazine oxidase

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Where can phase 1 enzymes generally be found?

ER:
- CYPs
- FMO
- ADH/ALDH
- Epoxide hydrolase
- (Esterases/ amidases)

Mitochondria:
- MAO A/B
- ADH/ALDH

Cytosol:
- Esterases/ amidases
- ADH/ALDH
- Epoxide hydrolase

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Which microsomal oxidation processes are involved in phase 1 reactions?

Aromatichydroxylation o Aliphatichydroxylation o Alicyclic
Heterocyclic
N-,S- and O-dealkylation o N-oxidation
N-hydroxylation
S-oxidation
Desulfaration
Deamination
Dehalogenation

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Which non-microsomal oxidation processes are involved in phase 1 reactions?

1) Amine oxidation
2) Alcohol and aldehyde oxidation
3) Peroxidases

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Describe the catalytic cycle of the cytochrome P-450 monooxygenase system

Addition of substrate to enzyme
Donation of an electron
Addition of oxygen and rearrangement
Donation of a second electron and loss of water

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What enzymes are involved in amine oxidations and what are their main characteristics?

Monoamine oxidases (MAO):
- Located in mitochondria
- Oxidative deamination of primary, secondary and tertiary amines

Diamine oxidases:
- Located in the liver and other tissue
- Soluble enzyme
- Is mainly involved in the metabolism of endogenous compounds such as the aliphatic diamine putrescine

Alcohol dehydrogenase:
- Is a cytosolic enzyme (soluble fraction)
- Located in the liver and also kidney and lungs
- Coenzyme NAD (sometimes NADP - slower)

Aldehyde dehydrogenase:
- Located in the mitochondria (ALDH2)

Other enzymes:
Molybdenum hydroxylases
Xanthine oxidase
Aldehyde oxidase

Peroxidases
- Are dependent on co-oxidation

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What enzymes are involved in reduction

Enzymes who catalyzes reduction:
* Gut bacterial azo- and nitro-reductase
* Cytochrome P-450 NADPH reductase –> works in low conditions of oxygen
* FAD alone (is not an enzyme)

(CYPs are also capable of reduction)

Reactions are dependent on:
- NAD(P)H

Inhibited by:
- O2

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What enzymes are involved in hydrolysis

1) Esterases
2) Amidases
3) Epoxide hydrolases
4) Peptidases
(CYPs are also capable of hydrolysis)

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Types of esterases

Class A)
A-esterases: Hydrolyze organophosphates (arylesterases, paraoxonase). These are not inhibited by phosphate triesters.

Class B)
B-esterases: Inhibited by organophosphates e.g. paraoxon (include carboxylesterases, cholinesterases, arylamidases)

Class C)
C-esterases: Are acetylesterases. These are not inhibited by paraoxon and the preferred substrates are acetyl esters.

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What are the steps and the product of glucuronidation (glucuronic acid conjugation)?

Conjugation with UDP-Glucuronic acid catalyzed by UDP-glucuronosyl transferase (UGT)

2) Glucuronic acid is afterwards converted into hydroxyl, carboxyl, nitrogen and sulfur

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Which enzyme catalyze sulfation

Sulfotransferase (SULT)
- with the cofactor 3’-phosphoadenosyl-5-phosphosulfate (PAPS):

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What are the product of sulfation?

Highly water soluble sulfate esters

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How can PAPS be depleted

When large amount of foreign compound conjugated with sulfate (e.g. paracetamol) is administered

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Which enzyme catalyze glutathione conjugation

Glutathione-S-transferases (GST)

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Describe the structure of glutathione

Glutathione is a tripeptide composed of:
1) Glutamat
2) Cysteine
3) Glycine

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Why is glutathione significant?

1) It has a reactive SH group 2) It is protected from protease digestion because the bond between the cysteine and glutamate is NOT a peptide bond 3) It is present in relatively high concentrations

How does glutathione detoxify?

It has a nucleophilic thiol group, and it can detoxify substances in one of three ways: 1. Conjugation catalyzed by a GSH transferase 2. Chemical reaction with a reactive metabolite to form a conjugate 3. Donation of a proton or hydrogen atom to reactive metabolites or free radicals, respectively

Acetylation is the major route of biotransformation for which compounds?

Sulfonamides, aromatic amines, hydrazines and hydrazides

Which enzymes catalyse acetylation reactions?

N-acetyltransferases: - NAT1; is in many tissues incl. red blood cells - NAT2; mostly in Kupffer cells in the liver but is also active in ER cells, in the spleen gut and lung

What is the purpose of acetylation?

To inactivate the reactive compound. Prevents further metabolization by CYP to more toxic metabolites (not like the conjugation reactions where the purpose is to make the compound more hydrophilic)

Which phase 2 reactions can paracetamol undergo?

Paracetamol can undergo: 1) Glucuronidation 2) Sulfation 3) Glutathione conjugation

What is the toxic intermediate of paracetamol

NAPQI

Which CYP enzyme converts paracetamol to it's intermediate?

CYP2E1 and CYP1A2

How is overdose on paracetamol treated? (antidote)

N-acetylcysteine (NAC) / (Methionin also) - Increases GSH synthesis leading to conjugation of NAPQI --> protect protein thiols - Increases sulfation by relieving saturation Overall result is reduced levels of NAPQI and oxidized protein thiols.

Where are the enzymes responsible for hydrolysis located?

1) Cytosol (cytosolic enzymes) 2) Microsomal (gut enzymes) 3) Blood

What are the substrates for hydrolysis?

Esters, thioesters, hydrazines, carbamates, peptides

Which groups are often reduced in vivo?

Aldehydes, ketones, disulfide, sulfoxide, quinone, N-oxide, alkenes, azo and nitro groups

Where is MAO located and which group of enzymes does it belong to?

MAO is located in the mitochondria, and it belongs to the group of enzymes called flavoproteins (FAD)

Which is the most abundant CYP in the liver?

CYP3A4

What is the main location of CYP and which co-factors are involved?

Location: ER Co-factor: NADPH

Where are FMO located and which co-factors are involved?

Location: ER Co-factor: FAD, NAD(P)H

Where are MAO located and which co-factors are involved?

Location: Mitochondira Co-factor: FAD, NAD(P)H

Where are the esterases / amidases located and which co-factors are involved?

Location: Cytosol, Plasma (ER) Co-factor: (H2O)

Where are alcohol dehydrogenase (ADH) / aldehyd dehydrogenase (ALDH) located and which co-factors are involved?

Location: Cytosol, ER, mitochondria Co-factor: FAD, NAD(P)H

Where are epoxide hydrolase located and which co-factors are involved?

Location: Cytosol, ER Co-factor: (H2O)

Which two enzymes metabolize a majority of large compounds?

CYP3A4 / CYP3A5 (CYP 3A5 are the garbage bins – metabolizes compounds that the others didn’t)

Which CYP enzyme metabolizes small, planar aromatics (amines)? (e.g. caffeine)

CYP1A1 and CYP1A2

Which CYP enzyme metabolizes acidic and lipophilic medium size substrates? (e.g. warfarin)

CYP2C9

Which enzymes likes nitrogen – amines and amides medium size? (e.g. omeprazol)

CYP2C19

Which CYP enzyme is highly active but cannot be induced and metabolize basic medium size substrates? (e.g. codeine)

CYP2D6

Which enzyme is considered the garbage bin – very open pocket – takes all of the compounds that none of the other takes (broad range) but is not specific? (e.g. paracetamol)

CYP3A4 and CYP3A5 (mostly CYP3A5)

Which mechanism of inductions are there?

Mechanism of induction are: 1. Increased synthesis of mRNA or precursor rRNA, 2. Increased stability of mRNA or rRNA 3. Decreased heme degradation 4. Decreased apoprotein degradation 5. Increased transport of RNA 6. Effects on DNA-dependent RNA polymerase

Which substrates induces the polycyclic hydrogen carbon and phenobarbital type CYPs respectively?

Planar substrates induces the polycyclic hydrogen carbon type (CYP1A1) Non-planar substrates induces the phenobarbital type (CYP2B1, CYP2B2 and CYP2B6A)

What is TCDD and how does it work?

TCDD is the most toxic compound (known of): 2,3,7,8-tetrachlorodibenzodioxin. The mechanism is receptor mediated induction of CYP1A1

Which CYP enzyme is induced by alcohol?

CYP2E1

What is the mechanism of induction of the CYP3A and CYP4A family?

It is receptor mediated induction by a chemical.

Which similarities and differences are there between CYPs and FMO (flavin-containing monooxygenase)?

- Both require NADPH, both are found in sER - FMO differes from CYP as it is thermolabile and metal free.

Where does glucuronide conjugation take place?

Where does sulfation / sulfate conjugation take place?

Cytosol

Where does gluathione conjugation take place?

Cytosol, mitochondria and nucleus

How does glutathione detoxify?

Where are the enzymes involved in xenobiotic metabolism produced?

Microsomes: CYP + UGT (UDP-glucuronosyl transferase) Cytosol: NAT (N-acetyltransferase) + GST (Glutathione-S-transferase)

Which reactions are catalyzed by CYPs?

Hydroxylation, epoxidation, (N, O, S)-dealkylations

What are required in CYP reactions?

O2, NAD(P)H and NADPH reductase