Metabolism of xenobiotics
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ENVR/TOXC 442 Fall 2012. Metabolism of Xenobiotics. II. Phase 1 Metabolism Aug 23, 2012 L.M. Ball Rosenau 158 [email protected] Phase I reactions. Chemical modification of xenobiotics Introduces or uncovers polar functional groups that provide sites for Phase II metabolism

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Metabolism of Xenobiotics

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Metabolism of xenobiotics

ENVR/TOXC 442 Fall 2012

Metabolism of Xenobiotics

II. Phase 1 Metabolism

Aug 23, 2012

L.M. Ball

Rosenau 158

[email protected]


Phase i reactions

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

Overview of oxidations, reductions, hydrolyses

  • Oxidation

    • Loss of electrons M M+ + e-

    • Gain of oxygen R + O RO


Oxidation reactions

Oxidation reactions

Hydroxylation


Metabolism of xenobiotics

Epoxidation


Metabolism of xenobiotics

Demethylation

Deethylation


Overview of oxidations reductions hydrolyses1

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

Reduction

  • Nitro to amino group

  • Chromium VI to Chromium III

Cr6+ + 3 e- Cr3+


Hydrolysis

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 + H2OR’-N-H + R-OH

      H H


Principal phase i enzymes

Principal Phase I enzymes

  • Cytochrome P450

  • Flavin monooxygenase

  • Monoamine oxidase

  • Esterases

  • Amidases

  • Hydrolases

  • Reductases, dehydrogenases, oxidases


Cytochrome p450

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 p4501

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

Overall reaction

R-H + O2 + NADPH + H+

R-OH + H2O + NADP+


Ferric protoporphyrin ix

Ferric protoporphyrin IX


Protoporphyrin ix

Protoporphyrin IX


Catalytic cycle of cytochrome p450

NADH

NADPH

Catalytic cycle of cytochrome P450

ROH

H+

Fe3+

+ RH

HO22-

Fe3+-RH

H2O

Fe3+-RH

+ e-

from NADPH-cytC reductase

H2O2

H+

HO2-

[Fe2+-RH]

Fe2+-RH

O2

[Fe2+-RH]

+O2

O2-.

H+ + e-


P450 and reductase in endoplasmic reticulum

P450 and reductase in endoplasmic reticulum


The p450 gene superfamily

The P450 gene superfamily

  • Format of nomenclature:

    CYPFamily/Subfamily/Gene

  • Family = 1, 2, …150 and counting

    • ~40% aa similarity

  • Subfamily = A, B,…H…W

    • 55-65% aa similarity

  • Gene = 1, 2..10 or above

    • >97% aa similarity (allelic variants)

  • Families grouped in Clans


Sub family family gene

Sub-Familyfamily Gene

CYP1 A1 BaP hydroxylation, O-deethyl’n

(PAC-inducible2 N-hydroxylation, O-deethylation

CYP2 A1 Testosterone 7-hydroxylation

2 Testosterone 15-hydroxylation

B1 Aliphatic hydroxylation

2 O-deethylation

C1 - 20+

2C19, mephenytoin hydroxylase


Metabolism of xenobiotics

Demethylation

Deethylation


Sub family family gene1

Sub-Familyfamily Gene

CYP1 A1 BaP hydroxylation, O-deethyl’n

(PAC-inducible2 N-hydroxylation, O-deethylation

CYP2 A1 Testosterone 7-hydroxylation

2 Testosterone 15-hydroxylation

B1 Aliphatic hydroxylation

2 O-deethylation

C1 - 20+

2C19, mephenytoin hydroxylase


Sub family family gene2

Sub- Familyfamily Gene

CYP2 D1 - 6+

2D6, debrisoquine hydroxylase

E1 C- and N-hydroxylation

small molecules

2

F1

CYP3 A1-4

3A4

CYP4 A1 Lauric acid - and

-1 hydroxylation


Metabolism of xenobiotics

Polymorphisms


Sub family family gene3

Sub-Familyfamily Gene

CYP11 (mito) A1 Steroid 11-hydroxylation

CYP17 A1 Steroid 17-hydroxylation

CYP21 A1 Steroid 21-hydroxylation

CYP51 A1 (Plants, yeast)

CYP52-66 AYeasts, fungi

CYP71-99, 701Plants

CYP101 A1 Pseudomonas putida P450cam

CYP102-132 ABacteria


Metabolism of xenobiotics

Contributions of enzymes to the metabolism of Pfizer-marketed drugs.

Guengerich, 2008.


Changes in p450 levels with age rats

Changes in P450 levels with ageRats

M: 2C6, 2C11, 3A2

F: 2A1, 2C6, 2C12

2A1

2C6

3A2


Metabolism of xenobiotics

  • Protein data bank 101

  • Human P450 nomenclature

  • Human P450s: substrates, inhibitors, inducers

  • Human CYP1A1 in Protein Knowledgebase


Flavin monooxygenase

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 reaction1

Overall reaction

R-H + O2 + NADPH + H+

R-OH + H2O + NADP+


Monoamine oxidase

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

Esterases

Hydrolyse esters to carboxylic acid and alcohol functional groups

Non-specific esterases in plasma, more substrate-specific forms in liver cytosol


Amidases

Amidases

Hydrolyse amides to carboxylic acids and amines (or ammonia)

Found in plasma and in liver cytosol


Hydrolases

Hydrolases

Hydrolyse ethers


Reductases dehydrogenases oxidases

Reductases, dehydrogenases, oxidases

In cytosol, endoplasmic reticulum, mitochondria


Outcomes

Outcomes

  • Slight gain in water solubility (ionizable hydroxyl, amino groups)

  • Sites for Phase 2 reactions – attachment of glucuronyl, sulfate, glutathione residues > major gain in water-solubility.

  • Formation of reactive electrophiles e.g. epoxides (especially polycyclic aromatic hydrocarbons)

  • Formation of sites for redox cycling (catechols <->quinones) > ROS


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