Metabolism of Xenobiotics - PowerPoint

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					      ENVR/TOXC 442 Fall 2009




Metabolism of Xenobiotics
    II. Phase 1 Metabolism
           Sept 1, 2009
            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

                               OH




Benzene               Phenol




          Hydroxylation
  Epoxidation                        O
          HC     CH2            HC       CH2




        Styrene            Styrene oxide



                       H


                           O
                                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
      NO2        NO        H NOH   H NH




• 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 + H2O       R’-N-H + R-OH
      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

        O                                O
                H2                                         H2
                           + H2O                           C
        C       C                        C
  H3C       O        CH3           H3C       OH   +   HO        CH3

    Ethyl acetate                   Acetic acid        Ethanol
                  Amidases

• Hydrolyse amides to carboxylic acids and
  amines (or ammonia)
• Found in plasma and in liver cytosol


     O                       O                    H
                 + H2O
             H
                                      +   H   N
 R   C   N               R   C
             H
                                 OH               H
                           Hydrolases
• Hydrolyse ethers


           H2       H2
                                             H2                 H2
           C        C          + H2O         C                  C
   H3C          O        CH3           H3C        OH   +   HO         CH 3

         Diethyl ether                   Ethanol            Ethanol
Reductases, dehydrogenases,
         oxidases

   • In cytosol, endoplasmic reticulum,
     mitochondria
            Alcohol dehydrogenase           Aldehyde dehydrogenase
        H
                  NAD+ NADH + H+        O
                                            NAD+ NADH + H+        O
  H3C   C    OH            H3C      C                   H3C   C
                                        H                            OH
        H


   Ethanol               Acetaldehyde                   Acetic acid
        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
                                                          CH 2
                                           CH 3 HC




                                                 N-

                  H3C                                            CH 3
     O
                                      N          Fe+3      N
             H2    H2
         C   C     C
                                                                 CH
OH
                                                                        CH 2
                                                 -
                                                 N



                         O
                                 H2
                             C   C        CH 2          CH 3

                    OH
Protoporphyrin IX
         Catalytic cycle of
         cytochrome P450

       H+         ROH
                             Fe3+    + RH
         HO22-      H2O
                                         Fe3+-RH
        Fe3+-RH                             + e- from NADPH-cytC
                        H+        H2O2           reductase
        HO2   -
                                           Fe2+-RH
       [Fe2+-RH]
                             O2      +O2
NADH
                         [Fe2+-RH]                      O
                                                          -.
                                                             2
  H+ + e-
NADPH
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-hydroxylation
                   2 Testosterone 15-hydroxylation

             B     1 Aliphatic hydroxylation
                   2 O-deethylation

             C     1 - 20+
                   2C19, mephenytoin hydroxylase
Demethylation
                 CH 3
             O
                                OH



                                                       H

                                         +     O   C
                                                           H


      Anisole                Phenol            Formaldehyde

Deethylation
             H2
             C CH3
         O                      OH


                                                       H
                                         +                     C   CH3
                                                       o

         N                      N
     H       C     CH3      H        C   CH3
         O                      O
    Phenacetin           Acetaminophen             Acetaldehyde
             Sub-
  Family     family            Gene

CYP1          A       1 BaP hydroxylation, O-deethyl’n
(PAC-inducible        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
       Sub-
Family family   Gene
CYP2     D      1 - 6+
                2D6, debrisoquine hydroxylase

         E      1 C- and N-hydroxylation
                      small molecules
                2
         F      1

CYP3     A      1-4
                3A4

CYP4     A      1 Lauric acid - and
                             -1 hydroxylation
       Sub-
Family family          Gene
CYP11 (mito) A   1 Steroid 11-hydroxylation

CYP17       A    1 Steroid 17-hydroxylation
CYP21       A    1 Steroid 21-hydroxylation

CYP51       A    1 (Plants, yeast)
CYP52-66    A    Yeasts, fungi
CYP71-99, 701    Plants
CYP101      A    1 Pseudomonas putida P450cam
CYP102-132 A     Bacteria
 Changes in P450 levels with age
             Rats
                    M: 2C6, 2C11, 3A2
2A1                 F: 2A1, 2C6, 2C12
2C6
3A2

				
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