Metabolism of Xenobiotics

II. Phase 1 Metabolism

Aug 30, 2011

L.M. Ball

Rosenau 158

lmball@unc.edu

ENVR/TOXC 442 Fall 2011

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

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

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

R’-O-R + H2O R’-O-H + R-OH

R’-N-R + H2O R’-N-H + R-OH

H 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

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

Sub-Family family Gene

CYP1 A 1 BaP hydroxylation, O-deethyl’n(PAC-inducible 2 N-hydroxylation, O-deethylation

CYP2 A 1 Testosterone 7-hydroxylation2 Testosterone 15-hydroxylation

B 1 Aliphatic hydroxylation2 O-deethylation

C 1 - 20+2C19, mephenytoin hydroxylase

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

Sub-Family family Gene

CYP1 A 1 BaP hydroxylation, O-deethyl’n(PAC-inducible 2 N-hydroxylation, O-deethylation

CYP2 A 1 Testosterone 7-hydroxylation2 Testosterone 15-hydroxylation

B 1 Aliphatic hydroxylation2 O-deethylation

C 1 - 20+2C19, mephenytoin hydroxylase

Sub- Family family Gene

CYP2 D 1 - 6+2D6, debrisoquine hydroxylase

E 1 C- and N-hydroxylationsmall molecules

2 F 1

CYP3 A 1-43A4

CYP4 A 1 Lauric acid - and -1 hydroxylation

Sub-Family family GeneCYP11 (mito) A 1 Steroid 11-hydroxylation

CYP17 A 1 Steroid 17-hydroxylationCYP21 A 1 Steroid 21-hydroxylation

CYP51 A 1 (Plants, yeast)CYP52-66 A Yeasts, fungiCYP71-99, 701 PlantsCYP101 A 1 Pseudomonas putida P450cam

CYP102-132 A Bacteria

Changes in P450 levels with ageRats

2A1

2C6

3A2

M: 2C6, 2C11, 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

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