1. Metabolism of Xenobiotics
Xiao Li

2. Introduction
Xenobiotics: is a compound that is foreign to the body ; is a chemical which is found in an organism but which is not normally produced or expected to be present in body.
Endogenous: Pigments , hormone
nonendogenous : Such as drugs , food additives, pollutants, toxin, etc
Most of these compounds are subject to metabolism (biotransformation) in human body.

3. Definition of the biotransformation
Conversion of lipophilic xenobiotics to water-soluble chemicals by a process catalyzed by enzymes in the liver and other tissues.
In most cases, biotransformation lessens the toxicity of xenobiotics, but many must undergo the process to exert their toxic effects.
卫生毒理学 3

4. Purpose of biotransformation
1. facilitates excretion: Converts lipophilic to hydrophilic compounds
2. Detoxification/inactivation: converts chemicals to less toxic forms
3. Metabolic activation: converts chemicals to more toxic active forms

5. Detoxification ≠ biotransformation
benzpyrene
3,4-Benzypyrene

6. Sites of biotransformation
Liver
Primary site! Rich in enzymes
Acts on endogenous and exogenous compounds
Extrahepatic metabolism sites
Intestinal wall
Sulfate conjugation
Esterase and lipases - important in prodrug metabolism
Lungs, kidney, placenta, brain, skin, adrenal glands

7. Knowledge of the metabolism of xenobiotics:
rational understanding of pharmacology and therapeutics , pharmacy, toxicology, cancer research , and drug addiction .

8. General Metabolic Pathways
Approximately 30 different enzymes catalyze reactions involved in xenobiotic metabolism; however, this note will only cover a selected group of them.
It is convenient to consider the metabolism of xenobiotics in two phases
phase Ⅰand phase Ⅱ

9. Phase I reactions ♣♣ Functionalization Purpose Oxidation Reduction
Hydrolytic reactions
Purpose
Introduction of polar functional groups in a molecules
♣ Increase a molecule’s polarity
♣ Does provide a site for phase II metabolism

10. Phase II reactions ♣♣ Conjugation ★ Purpose
Introduce highly polar conjugates:
☻☻Glucuronic acid ☻☻ Sulfate
Detoxification
Glycine or other Amino Acids (some solubility), Acetyl , Methylations , Glutathione
★ Site of attachment often introduced in Phase I
Hydroxyl , Carboxylate , Amino

11. Comparing Phase I & Phase II

12. RH + O2 + NADPH + H+  R-OH + H2O + NADP+
Phase Ⅰ: Oxidation
1. Hydroxylation
RH + O2 + NADPH + H+  R-OH + H2O + NADP+
Addition of an oxygen atom or bond
Require NADH or NADPH and O2 as cofactors
RH: drugs, cacinogens, pesticides, petroleum products, pollutants, steroids, eicosanoids, fatty acids, retinoids, etc.
Enzyme:
Cytochrome P450s-dependent monooxygenase

13. Hydroxylation：O2
   * It has been shown by the use of O2 that one atom of oxygen enters R-OH and one atom enters H2O.
* This dual fate of the oxygen accounts for the former naming of monooxygenases as “mixed-function oxidases”.
RH + O2 + NADPH + H+  R-OH + H2O + NADP+

14. Cytochrome P450s-dependent monooxygenase
-----the most versatile biocatalyst
----Works on a large number of diverse compounds
★ Microsomal drug oxidations require
cytochrome P450,
cytochrome P450 reductase,
NADPH , & O2
The actual reaction mechanism is as follows:

16. Cytochrome P450s-dependent monooxygenase
CYP or Cytochrome P-450
★ Heme proteins
★ Iron containing porphyrin - binds O2
★ The name cytochrome P450 is derived from the spectral properties of this hemoprotein  in its reduced (ferrous, Fe2+) form, it binds CO to give a complex that absorbs light maximally at 450 nm

17. Cytochrome P-450 Enzymes isolated by disruption of the liver cells
Endoplasmic reticulum - microsomes when disrupted
Enzymes are membrane bound
Explains why lipophilic drugs are processed
Catalytic process  heme binds O2

18. Cytochrome P450: Isozymes
★ Isozymes - multiple forms of an enzyme
★ Supergene family
- More than 8,000 P450 genes as of November/2007
- More than 368 gene families, 814 subfamilies
- Human: 18 families, 43 subfamilies, 57 sequenced
genes
★ Nomenclature
CYP1A2
family
subfamily
individual member of that subfamily

19. Cytochrome P450
This enzyme is very important. approximately 50% of the drugs that humans ingest are metabolized by isoforms of cytochrome P450.
These enzymes also act on various carcinogens and pollutants.

20. Substrates
Allyl
Vinyl
Benzyl
Aromatic
Alicyclic
Aliphatic

21. 2. Monoamine oxidase, MAO RCH2NH2+O2+H2O2 RCHO+NH3+H2O
★ MAO catalyze the oxidative deamination of monoamines.
★ Oxygen is used to remove an amine group from a molecule, resulting in the corresponding aldehyde and ammonia.
★ MAO are found bound to the outer membrane of mitochondria in most cell types in the body. They belong to protein family of flavin containing amine oxidoreductases.

22. 3. ADH and ALDH
ADH alcohol dehydrogenase
ALDH aldehyde dehydrogenase
Alcohol Dehydrogenase belongs to the oxidoreductase family of enzymes.
high concentrations within the liver and kidney.

23. Function
The primary and most common role of ADH in humans is to detoxify incoming ethanol by converting it into aldehyde.
The resulting aldehyde, a more toxic molecule than ethanol, is quickly converted into acetate by aldehyde dehydrogenase (ALDH) and other molecules easily utilized by the cell.
ALDH
ADH

24. ADH
ALDH
During this reaction, hydrogen is removed from the alcohol and transferred to a molecule called nicotinamide adenine dinucleotide (NAD), converting it to reduced NAD (NADH).
NADH participates in numerous other metabolic reactions, passing on the hydrogen to other compounds or electron transfer chain.

26. Absorption Soluble in water
stomach
20%
Soluble in water
Small size - penetrates everywhere, easily crosses all bio membranes
Rapidly absorbed from GI
small
intestine
80%
Blood
until metabolized

27. In people who consume alcohol at moderate levels and/or only occasionally, most of the alcohol is broken down by ADH and ALDH.
after higher alcohol consumption, The MEOS plays a role in alcohol metabolism.
CH3CH2OH + NADPH + O2 + H CH3CHO + NADP+ + 2H2O
MEOS
CH3CHO
ALDH
CH3COOH
MEOS: Microsomal Ethanol-Oxidizing System , is also called Cytochrome P450-dependent Microsomal Ethanol Oxidizing System. converts alcohol to acetaldehyde

28. CH3CH2OH + NADPH + O2 + H+ CH3CHO + NADP+ + 2H2O
MEOS
This reaction also relies on oxygen and NADPH, and results in the formation of NADP and water.
◆consume oxygens of liver and NADPH
◆ As byproducts of these reactions, oxygen radicals or reactive oxygen species (ROS) are generated. These ROS can contribute to liver damage through a variety of mechanisms.

29. ADH and MEOS consume energy ADH MEOS In liver cytoplasma microsome
substrate
alcohol、NAD+
alcohol 、NADPH、O2
Km to alcohol
2mmol/L
8.6mmol/L
Induced by alcohol no
yes
energy
Release energy
consume energy

30. Although the rate at which ADH breaks down alcohol generally stays the same, the activity of the MEOS can be increased (induced) by alcohol consumption.

31. Because the MEOS metabolizes not only alcohol but also other compounds (certain medications), enhanced MEOS activity resulting from high alcohol consumption also can alter the metabolism of those medications.
This may contribute to harmful interactions between alcohol and those medications or otherwise influence the activity of those medications.

32. Alcoholism leads to fat accumulation in the liver, hyperlipidemia, and ultimately cirrhosis.

33. 4. Nitro and Azo Reduction
Phase Ⅰ: Reduction
4. Nitro and Azo Reduction
NADPH dependent microsomal and nitro-reductase enzymes.
Bacterial reductases play a role in enterohepatic recirculation of nitro or azo containing drugs.

34. 2005 , sudan red incident
chili patse
sudan red

35. nitroreductase
chloromycetin

36. 5. Hydrolysis Substrates: esters , amide , glycoside, etc.
Phase Ⅰ: Reduction
5. Hydrolysis
Substrates: esters , amide , glycoside, etc.
Catalyzed by widely distributed hydrolytic enzymes
Hydrolysis of esters  major metabolic pathway for ester drugs
☻Non-specific esterases (liver, kidney, and intestine)
☻Plasma pseudocholinesterases also participate
esterase
Acetylsalicylic Acid, ASA
salicylic acid

37. Phase II: Conjugation
In phase Ⅰ reactions, xenobiotics are generally converted to more polar, hydroxylated derivatives.
In phase Ⅱ reactions, these derivatives are conjugated with molecules such as glucuronic acid, sulfate, or glutathione.
This renders them even more water-soluble, and they are eventually excreted in the urine or bile.

38. xenobiotic excrection nontoxic metabolite cancer Phase I Phase II
Protection
Elimination
Reactive
metabolite
Cell injury
Antibody product
mutation
cancer

39. Five types of phase II reactions
A.      Glucuronidation
B.      Sulfation
C. Conjugation with glutathione
D. Acetylation
E. Methylation

40. 1. Glucuronidation the most frequent conjugation reaction.
UDP-glucuronic acid (UDPGA) is the glucuronyl donor
UDP-glucuronyl transferases (UGT), present in both the endoplasmic reticulum(ER) and cytosol, are the catalysts.
Liver, lung, kidney, skin, brain and intestine
Attachment sites are hydroxyls
Alcohols, phenols, enols, N-hydroxyls, acids
Oxygen site often from Phase I

41. Oxygen glucuronides cont…
Alcohol hydroxyl example N H Cl O 2 C
Chloramphenicol - Chloromycetin -
Phenol hydroxyl example

43. 2. Sulfate Conjugation Catalyzed by sulfotransferases
Some alcohols, arylamines, and phenols are sulfated.
Catalyzed by sulfotransferases
liver, kidney and intestine
Sulfate donor: adenosine 3’-phosphate-5’-phosphosulfate (PAPS); this compound is called “active sulfate.”
Leads to inactive water-soluble metabolites
Glucuronate conjugation often more competitive process
sulfotransferase
PAPS

44. Sulfate Conjugation
+PAP
estrone sulfate
estrone
＋PAPS

45. 3. Conjugation with glutathione
glutamic acid, cysteine, glycine
R + GSH R-S-G
GST
where R= an electrophilic xenobiotics
R: epoxides and halogenides
GST: Glutathione S-Transferases (Liver and kidney)

46. Glutathione (GSH) Conjugation
DETOXIFICATION of electrophiles!
Electrophilic chemicals cause:
Tissue necrosis
Carcinogenicity
Mutagenicity
Teratogenicity
The thiol (SH group) ties up potent electrophiles

47. Glutathione S-transferase
(+)benzo[a]pyrene-
7,8-dihydrodiol-
9-10-epoxide
glutathione HO HO
GST
+ glutathione OH
DNA reactive;
lung and skin tumors
Inactive
DETOXIFICATION

48. Glutathione (GSH) Conjugation
aflatoxin b-1 epoxide

49. 4. Acetylation X + Acetyl-CoA - - - - >Acetyl-X + CoS
     where X represents a xenobiotics.
(for: aromatic amines)
Enzyme: acetyltransferases
present in the cytosol of various tissues, particularly in liver.
isoniazid

50. Important for drugs with primary amino groups
sulfanilamide
Important for drugs with primary amino groups
Generally, metabolites are nontoxic and inactive
Acetylation does NOT increase water solubility
Detoxification or termination of drug activity

51. Methylation A few xenobiotics are subject to
methylation by methyltransferase,
emplyoing S-adenosylmethione(SAM) as the methyl donor.
SAM
catechol

52. Metabolism via Methylation
Key for biosynthesis of many compounds
Important in the inactivation of physiologically active biogenic amines  neurotransmitters
norepinephrine, dopamine, serotonin, histamine
Minor pathway in the metabolism of drugs
Methylation does NOT increase water solubility
Most methylated products are inactive

53. Factors that influence metabolism
Age
older people less efficient at metabolism
Sex
Linked to hormonal differences
Heredity
Genetic differences can influence amounts and efficiency of metabolic enzymes
Disease states
Liver, cardiac, kidney disease

54. Summary Phase I reactions: Phase II: Conjugation Rx
Xenobiotic, Biotransformation
Phase I reactions:
Purpose:
Enhances elimination
Converts chemical to less toxic forms (detoxification)
Converts chemicals to more toxic active forms (activation)
Functionalization:
Oxidation: monooxygenase, CYP450
Reduction: ADH, ALDH
Hydrolytic reactions: esterase
Phase II: Conjugation Rx
Purpose: more water-soluble, excreted in the urine or bile
Glucuronidation, Sulfation, Conjugation with glutathione, Acetylation, Methylation

55. ALCOHOL Mechanism of Fatty Liver
The likelihood of hypoglycemia is also increased in alcoholics when they fast, as they often have low hepatic stores of glycogen because of poor nutrition.
The shift in the NADH/NAD+ ratio also inhibits β-oxidation of fatty acids and promotes triglyceride synthesis; this increases hepatic synthesis of VLDL, and the remaining excess triglyceride is deposited in the liver.