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