ENVR/TOXC 442 Fall 2011
Metabolism of Xenobiotics
II. Phase 1 Metabolism
Aug 30, 2011
L.M. Ball
Rosenau 158
lmball@unc.edu
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
Epoxidation
HC
O
HC
CH2
Styrene
CH2
Styrene oxide
H
O
H
Benzo[a]pyrene
Benzo[a]pyrene
7,8-oxide
Demethylation
CH3
O
OH
H
+
O
C
H
Anisole
Phenol
Formaldehyde
Deethylation
H2
C CH3
O
OH
H
+
C
CH3
o
N
H
N
C
CH3
O
Phenacetin
H
C
CH3
O
Acetaminophen
Acetaldehyde
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
NO2
NO
HNOH
• Chromium VI to Chromium III
Cr6+ + 3 e-
Cr3+
H NH
Hydrolysis
• Addition of water
– Cleavage of R-O or R-N bond accompanied
by addition of H2O
R’-O-R + H2O
R’-N-R + H2O
H
R’-O-H + R-OH
R’-N-H + R-OH
H
Principal Phase I enzymes
•
•
•
•
•
•
•
Cytochrome P450
Flavin monooxygenase
Monoamine oxidase
Esterases
Amidases
Hydrolases
Reductases, dehydrogenases, oxidases
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
CH2
CH3 HC
NCH3
H3C
O
C
H2
C
N
H2
C
Fe+3
N
CH
OH
CH2
N
O
C
OH
H2
C
CH2
CH3
Protoporphyrin IX
Catalytic cycle of
cytochrome P450
ROH
H+
Fe3+
HO22-
+ RH
H2O
Fe3+-RH
HO2
-
[Fe2+-RH]
NADH
H+ + eNADPH
H2O2
H+
Fe3+-RH
+ e- from NADPH-cytC
reductase
Fe2+-RH
O2
+O2
[Fe2+-RH]
-.
O
2
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
Family
Subfamily
Gene
CYP1
A
(PAC-inducible
1 BaP hydroxylation, O-deethyl’n
2 N-hydroxylation, O-deethylation
CYP2
A
1 Testosterone 7-hydroxylation
2 Testosterone 15-hydroxylation
B
1 Aliphatic hydroxylation
2 O-deethylation
C
1 - 20+
2C19, mephenytoin hydroxylase
Demethylation
CH3
O
OH
H
+
O
C
H
Anisole
Phenol
Formaldehyde
Deethylation
H2
C CH3
O
OH
H
+
C
CH3
o
N
H
N
C
CH3
O
Phenacetin
H
C
CH3
O
Acetaminophen
Acetaldehyde
Family
Subfamily
Gene
CYP1
A
(PAC-inducible
1 BaP hydroxylation, O-deethyl’n
2 N-hydroxylation, O-deethylation
CYP2
A
1 Testosterone 7-hydroxylation
2 Testosterone 15-hydroxylation
B
1 Aliphatic hydroxylation
2 O-deethylation
C
1 - 20+
2C19, mephenytoin hydroxylase
SubFamily family
CYP2
Gene
D
1 - 6+
2D6, debrisoquine hydroxylase
E
1 C- and N-hydroxylation
small molecules
2
1
F
CYP3
A
1-4
3A4
CYP4
A
1 Lauric acid - and
-1 hydroxylation
SubFamily family
Gene
CYP11 (mito) A
1 Steroid 11-hydroxylation
CYP17
CYP21
1 Steroid 17-hydroxylation
1 Steroid 21-hydroxylation
A
A
CYP51
A
CYP52-66
A
CYP71-99, 701
CYP101
A
CYP102-132 A
1 (Plants, yeast)
Yeasts, fungi
Plants
1 Pseudomonas putida P450cam
Bacteria
Changes in P450 levels with age
Rats
M: 2C6, 2C11, 3A2
2A1
2C6
3A2
F: 2A1, 2C6, 2C12
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
O
O
C
H3C
O
H2
C
Ethyl acetate
+ H2O
CH3
H2
C
C
H3C
OH
Acetic acid
+
HO
CH3
Ethanol
Amidases
• Hydrolyse amides to carboxylic acids and
amines (or ammonia)
• Found in plasma and in liver cytosol
O
H
R
C
N
H
O
+ H2O
R
H
+
C
OH
H
N
H
Hydrolases
• Hydrolyse ethers
H2
C
H3C
H2
C
O
Diethyl ether
H2
C
+ H2O
CH3
H3C
H2
C
OH
Ethanol
+
HO
Ethanol
CH3
Reductases, dehydrogenases,
oxidases
• In cytosol, endoplasmic reticulum,
mitochondria
Alcohol dehydrogenase
H
H3C
C
Aldehyde dehydrogenase
NAD + NADH + H+
OH
H3C
O
NAD + NADH + H+
C
H3C
H
O
C
OH
H
Ethanol
Acetaldehyde
Acetic acid