metabolism - biotransformation supplementary readings: casarett and doull,chapter 6 timbrell,...
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Metabolism - Biotransformation
Supplementary readings: Casarett and Doull,Chapter 6
Timbrell, Chapters 4 and 5
Non-polar(lipophilic)
Hydrophobic
Lipophobic Hydrophilic(Polar)
XENOBIOTIC
INTERMEDIATE METABOLITE
ELIMINATION
WATER-SOLUBLE METABOLITE
May be reactive/toxic
Can accumulate in tissues
Phase I MetabolismOxidation
Phase II Metabolism
ConjugationSol
ubili
ty in
lipi
ds
Sol
ubili
ty in
wat
er
What is a xenobiotic ?
Phase I reactions
• Chemical modification of xenobiotics
• Introduces or uncovers polar functional groups that provide sites for Phase II metabolism
• Major classes of reaction:– Oxidation– Reduction– Hydrolysis
Overview of oxidations, reductions, hydrolyses
• Oxidation
–Loss of electrons M M+ + e-
–Gain of oxygen R + O RO
Oxidation reactions
Hydroxylation
OH
Benzene Phenol
Styrene Styrene oxide
HC CH2 HC CH2
OEpoxidation
O
H
H
Benzo[a]pyrene Benzo[a]pyrene 7,8-oxide
Overview of oxidations, reductions, hydrolyses
• Reduction
– Gain of electrons M+ + e- M
– Loss of oxygen RO R + O
– Gain of hydrogen R + H RH
Reduction
• Nitro to amino group
• Chromium VI to Chromium III
NO NOHNO2 NHH H
Cr6+ + 3 e- Cr3+
Hydrolysis
• Addition of water– Cleavage of R-O or R-N bond accompanied by addition
of H2O
Ether
Amide
R
O
R'
+ H2O R OH HO R'+
R
N
R'
+ H2O R N HO R'+
H
H
H
Principal Phase I enzymes
• Cytochrome P450
• Flavin monooxygenase
• Monoamine oxidase
• Esterases
• Amidases
• Hydrolases
• Reductases, dehydrogenases, oxidases
Flavin monooxygenase
• Flavoprotein
• Mixed-function amine oxidase
• Located in smooth endoplasmic reticulum, in human, pig, rabbit liver, guinea-pig lung, human kidney
• Uses NADPH as a source of reducing equivalents
• Not inducible
Overall reaction
R-H + O2 + NADPH + H+
R-OH + H2O + NADP+
Monoamine oxidase
• Metabolizes endogenous monoamine neurotransmitters
• Uses NADPH as a source of reducing equivalents
• Found in the endoplasmic reticulum and in mitochondria, of nerve endings and liver
Esterases
• Hydrolyse esters to carboxylic acid and alcohol functional groups
• Non-specific esterases in plasma, more substrate-specific forms in liver cytosol
H3CC
O
H2C
CH3
O
H3CC
O
OH HO
H2C
CH3+
Ethyl acetate Acetic acid Ethanol
+ H2O
Amidases
• Hydrolyse amides to carboxylic acids and amines (or ammonia)
• Found in plasma and in liver cytosol
R C
O
N
H
HR C
O
OH
+ H N
H
H
+ H2O
Hydrolases
• Hydrolyse ethers
H3C
H2C
O
H2C
CH3 H3C
H2C
OH HO
H2C
CH3+
+ H2O
Diethyl ether Ethanol Ethanol
Reductases, dehydrogenases, oxidases
• In cytosol, endoplasmic reticulum, mitochondria
H3C C OH
H
H
H3C C
O
H
H3C C
O
OH
Ethanol Acetaldehyde Acetic acid
NAD+NADH + H+
NAD+ NADH + H+
Alcohol dehydrogenase Aldehyde dehydrogenase
Cytochrome P450
• Heme protein
• Terminal oxidase of the mixed-function oxidase (MFO) electron-transfer system
• Located in the smooth endoplasmic reticulum of all major organs and tissues
• Uses NADPH as a source of reducing equivalents
• Inducible
Cytochrome P450
• Heme protein
• Terminal oxidase of the mixed-function oxidase (MFO) electron-transfer system
• Located in the smooth endoplasmic reticulum of all major organs and tissues
• Uses NADPH as a source of reducing equivalents
• Inducible
Overall reaction
R-H + O2 + NADPH + H+
R-OH + H2O + NADP+
Ferric protoporphyrin IX
N
N-
N
-N
Fe+3
CH3 HCCH2
H3C
CH3
CH3
CH
CH2
CH2
H2CC
O
OH
H2C
H2CC
O
OH
Protoporphyrin IX
Catalytic cycle of cytochrome P450
from NADPH-cytC reductase
Fe3+
Fe3+-RH
Fe2+-RH
O2
[Fe2+-RH]
HO2-
[Fe2+-RH]
HO22-
Fe3+-RH
+ RH
+ e-
+O2
ROH
H+ + e-
NADPH
NADH
H2O
H2O2H+
H+
O2
-.
P450 and reductase in endoplasmic reticulum
The P450 gene superfamily• Format of nomenclature:
CYPFamily/Subfamily/Gene• Family = 1, 2, …150 and counting
– ~40% aa similarity• Subfamily = A, B,…H…
– 55-65% aa similarity• Gene = 1, 2..10 or above
– >97% aa similarity (allelic variants)• Families grouped in Clans• http://www.cypalleles.ki.se/
Major CYP450 isoformsFamily Subfamily Individual
CYP1 A1 BaP hydroxylation, O-deethylation
2 N-hydroxylation, O-deethylation
CYP2
A1 Testosterone 7α-hydroxylation
2 Testosterone 15α-hydroxylation
B1 Aliphatic hydroxylation
2 O-deethylation
C1-20+
CYP2C19, mephenytoin hydroxylase
D1-6+, dextromethorphan O-demethylation
CYP2D6, debrisoquine hydroxylase
E 1 C and N hydroxylation, small molecules
CYP3 A1 – 4
CYP3A4 major form in human liver
Demethylation
Deethylation
O
N
H2C CH3
H C CH3
O
OH
N
H C CH3
O
+
Phenacetin Acetaminophen
C CH3
o
H
Acetaldehyde
O
CH3
OH
CO
H
H
+
Anisole Phenol Formaldehyde
More CYP450 isoformsFamily Subfamily Individual
CYP4 A1 Lauric acid ω- and
ω-1 hydroxylation
CYP11(mitochondria)
A 1 Steroid 11β-hydroxylation
CYP17 A 1 Steroid 17α-hydroxylation
CYP21 A 1 Steroid 21β-hydroxylation
CYP51 Plants. yeasts
CYP52-66 Yeasts, fungi
CYP71-99, 701+ Plants
CYP101 A 1 Pseudomonas putida P450cam
CYP102-132 Bacteria
Induction
• Increased transcription
• Increased protein synthesis
• Enhanced stability of protein
• Synthesis of enzyme with higher catalytic activityInducible forms of CYP: CYP1A1 (PAH), CYP2B, CYP3A4 (PB), CYP2E1 (EtOH)
Constitutive: CYP2A
http://medicine.iupui.edu/flockhart/table.htm
Example:
Ah-locus mediated inductionAhR, receptor in cytoplasm, binds ligand: eg PAHs, TCDD, some PCBs
Bound AhR loses 2 heat-shock proteins (hsp90), becomes phosphorylated
Activated bound AhR migrates to nucleus, forms complex with Ah receptor nuclear translocation factor Arnt
AhR-Arnt complex binds to regulatory sequences in DNA (DRE, dioxin-responsive elements)
Transcription of CYP1A1 gene and other genes
Other “inducers” also interact with receptors
• CAR, responds to phenobarbital-type inducers, regulates CYP2B, CYP3A4, CYP reductase, transferases (?)
• PXR, CYP3A
• PPARα, CYP4A
• LXR, FXR control enzymes involved in bile acid and lipid metabolism
The Official Cytochrome p450 Webpage: REAL ULTIMATE POWER
The Official Cytochrome p450 Webpage. p450 BM-3 crystal structure. Real Ultimate Power. Hi, this site is all about cytochromes p450, REAL CYTOCHROMES p450. ...www.its.caltech.edu/~atobias/RUP-p450.html - 19k
http://www.google.com/search?q=cache:LX1MqRE52G8J:www.its.caltech.edu/~atobias/RUP-p450.html+Cytochrome+P450&hl=en&ct=clnk&cd=22&gl=us&client=firefox-a
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