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 lmball@unc.edu. 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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ENVR/TOXC 442 Fall 2012

Metabolism of Xenobiotics

II. Phase 1 Metabolism

Aug 23, 2012

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


Demethylation

Deethylation


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

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 + H2OR’-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


Protoporphyrin IX


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


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-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


Demethylation

Deethylation


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- 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


Polymorphisms


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


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

Guengerich, 2008.


Changes in P450 levels with ageRats

M: 2C6, 2C11, 3A2

F: 2A1, 2C6, 2C12

2A1

2C6

3A2


  • Protein data bank 101

  • Human P450 nomenclature

  • Human P450s: substrates, inhibitors, inducers

  • Human CYP1A1 in Protein Knowledgebase


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


Amidases

Hydrolyse amides to carboxylic acids and amines (or ammonia)

Found in plasma and in liver cytosol


Hydrolases

Hydrolyse ethers


Reductases, dehydrogenases, oxidases

In cytosol, endoplasmic reticulum, mitochondria


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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