1. Metabolism of Xenobiotics
ENVR/TOXC 442 Fall 2011
Metabolism of Xenobiotics
II. Phase 1 Metabolism
Aug 30, 2011
L.M. Ball
Rosenau 158

2. 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

3. Overview of oxidations, reductions, hydrolyses
Loss of electrons M M+ + e-
Gain of oxygen R + O RO

4. Oxidation reactions
Hydroxylation

5. Epoxidation

6. Demethylation
Deethylation

7. Overview of oxidations, reductions, hydrolyses
Gain of electrons M+ + e- M
Loss of oxygen RO R + O
Gain of hydrogen R + H RH

8. Reduction Cr6+ + 3 e- Cr3+ Nitro to amino group
Chromium VI to Chromium III
Cr e Cr3+

9. 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

10. Principal Phase I enzymes
Cytochrome P450
Flavin monooxygenase
Monoamine oxidase
Esterases
Amidases
Hydrolases
Reductases, dehydrogenases, oxidases

11. 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

12. 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

13. Overall reaction
R-H + O2 + NADPH + H+
R-OH + H2O + NADP+

15. Ferric protoporphyrin IX

16. Protoporphyrin IX

17. 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
NADPH
NADH
O2-.
H+ + e-

19. P450 and reductase in endoplasmic reticulum

20. 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, or above
>97% aa similarity (allelic variants)
Families grouped in Clans

21. Sub- Family family Gene
CYP A 1 BaP hydroxylation, O-deethyl’n
(PAC-inducible 2 N-hydroxylation, O-deethylation
CYP A 1 Testosterone 7-hydroxylation
2 Testosterone 15-hydroxylation
B 1 Aliphatic hydroxylation
2 O-deethylation C
2C19, mephenytoin hydroxylase

22. Demethylation
Deethylation

23. Sub- Family family Gene
CYP A 1 BaP hydroxylation, O-deethyl’n
(PAC-inducible 2 N-hydroxylation, O-deethylation
CYP A 1 Testosterone 7-hydroxylation
2 Testosterone 15-hydroxylation
B 1 Aliphatic hydroxylation
2 O-deethylation C
2C19, mephenytoin hydroxylase

25. Sub- Family family Gene
CYP2 D
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

27. Sub- Family family Gene
CYP11 (mito) A 1 Steroid 11-hydroxylation
CYP A 1 Steroid 17-hydroxylation
CYP A 1 Steroid 21-hydroxylation
CYP A 1 (Plants, yeast)
CYP A Yeasts, fungi
CYP71-99, 701 Plants
CYP A 1 Pseudomonas putida P450cam
CYP A Bacteria

29. Changes in P450 levels with age Rats
M: 2C6, 2C11, 3A2
F: 2A1, 2C6, 2C12
2A1
2C6
3A2

30. 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

31. Overall reaction
R-H + O2 + NADPH + H+
R-OH + H2O + NADP+

32. 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

33. Esterases
Hydrolyse esters to carboxylic acid and alcohol functional groups
Non-specific esterases in plasma, more substrate-specific forms in liver cytosol

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

35. Hydrolases
Hydrolyse ethers

36. Reductases, dehydrogenases, oxidases
In cytosol, endoplasmic reticulum, mitochondria