Cytochrome P450: the importance of this enzyme in xenobiotic metabolism

1. Cytochrome P-450: The
Importance of this Enzyme in
Xenobiotic Metabolism
Xenobiotic Metabolism EOH

2. Introduction to CYP450
• Hemeproteins/ Heme-thiolate proteins: form complexes with CO
(absorbance at 450nm)
• Bio-activation and detoxification of xenobiotics
• Synthesis of biologically active compounds: steroids, prostaglandins,
arachidonate metabolites
• Localized in smooth ER of hepatocytes and cells of intestine
• Humans have 57 genes and >57 59 pseudogenes subcategorized into
18 families and 44 sub-families

4. Contribution of CYPs to Xenobiotic
Metabolism in Humans
Sub-family Chemicals Metabolised
CYP1A Caffeine, phenacetin, theophylline, melatonin,pyrethroids, clozapine,
tizanidine, herbicides, insecticides
CYP2A Nicotine, pesticides like carbaryl, imidacloprid, DEET, diuron,
carbosulfan
CYP2B Cyclophosphamide, bupropion, diazepam, ifosamide, efavirenz
CYP2C Tolbutamide, glipizide, fluvastatin, phenytoin, pyrethroid insecticides
CYP2D Metoprolol, timolol
CYP2E Acetaminophen, enflurane, halothane, atrazine, carbaryl, parathion,
diuron
CYP3A Triazolam, simvastatin, atorvastatin, quinidine, carbamate,
phosphorothioate, neonicotinoid insecticides

5. Mechanism of Action
• Hydroxylation reactions, especially oxidation reactions:
Other Oxidation Reactions
Microsomal
oxidation
N
Dealkylation:
morphine to
normorphine
S dealkyation:
6
methylthiopuri
ne to 6
mercaptopurin
e
O
dealkylation:
phenacetin
to
paracetamol
N oxidation:
Dpsone,
CPM
S Oxidation:
CPZ to CPZ
sulfoxide
Deamination:
amphetamine
to
phenylaceton
e
Desulfurisati
on:
parathion to
paraoxon

6. Cytochrome P450 Cycle in Drug Oxidation

7. Introduction to Xenobiotics
• Xenobiotic is a foreign compound within any organism
• Reactive species of xenobiotics can bind to protein changing their
antigenicity, called hapten
• These can interact with DNA making it is important in chemical carcinogenesis
stuies
• Examples: drugs, food additives, carcinogens, pollutants, insecticides,
etc
• Enter mainly through medication and food via intestine, lungs or skin

8. Toxic Effects
Xenobiotic
Non-toxic
Metabolite
Reactive
Metabolite
Excretion
Phase I
Phase II
Protection
Elimination
Cell Injury Antibody
product;
Cell Injury
Mutation

9. Entry and Exit
Entry
Simple
Diffusion
Facilitated
Diffusion
Active
Transport
Endocytosis
Exit
Primary
Active
transport
Special
ATPases: ABC
(ATP binding
cassettes)
MRP (multidrug
resistant
proteins)

10. Xenobiotic Biotransformation
• Foreign lipophilic compounds get converted to hydrophilic
metabolites
• Purpose:
• Facilitate excretion
• Detoxification/inactivation
• Metabolic activation
• Elimination of xenobiotics after enzymatic catalysis:
• More polar (urine, bile, stool, sweat)
• Volatile substance by lungs
• Excretion into human milk
• Intestinal deconjugation

11. Metabolism
• Elimination is essential to remove toxins accumulated due to the
foods we ate
• Food broken down in stomach, absorbed by small intestine ferried to
liver
• Liver detoxifies it before it enters the circulatory system
• It accomplishes this by two main types of metabolisms that deal with
xenobiotics and one dealing wit their transport
• Phase I, II and III

12. Phase I Metabolism
- Small chemical changes to make compound more hydrophilic
- Easy elimination by kidneys
- Addition or revealing OH-, amine, sulfhydryl, hydrophilic groups
- Hydroxylation (major), oxidation, reduction reactions

13. Example of Hydroxylation

14. Other Enzymes
• Flavin-containing monooxygenases: oxidize nucleophilic N, S, P
• Non-inducible
• Present in microsomal fraction of liver, kidney, lung
• Examples: nicotine to nicotine-1-N-oxide, 2-AAF
carcinogen to N-hydroxyl-2-AAF
• Epoxides: highly mutagenic and reactive compo-
unds, converted by epoxide hydrolase into dihyd-
rodiols
• Forms: microsomal, soluble, cholesterol, LTA4, Hepoxilin
• Epoxide hydrolase can detoxify aflatoxin-epoxide by DNA binding

15. Non-microsomal Reactions
• Oxidative deamination of mono-amines
• Oxygen removes amine from a molecule resulting in aldehyde or ammonia
• Belong to Flavin containing amine oxidoreductases family
RCH2NH2 + O2 + H2O2 RCHO + NH3 + H2O
• Hydrolysis
• Hydrolysis of esters and amides
• Mediated by liver microsomal amidases, esterases, deacylases
• Example: formation of Acetylsalicylic acid from ASA by esterase

16. Phase II Reactions
• When Phase I reactions are insufficient for xenobiotic elimination or
when it produces a reactive metabolite
• Addition of larger polar group like glucuronide, in order to increase
compound solubility
• Main reactions are transferase reactions
• Examples: UG transferse, N-acetyl transferase, GST, sulphotransferase

17. Glucuronidation
• UDP-glucuronic acid acts as donor
• Glucuronide maybe attached to oxygen, nitrogen, sulfur groups

18. Biotransformation of amphetamine

19. Other Reactions
• Sulfation of alcohols, arylamines, pheols, steroids, glycosainoglycans,
glycolipids, glycoproteins
• Adenosine 3- phosphate-5- phosphosulfate (PAPS) acts as donor
• Results in inactive water soluble molecules
• Glutathione: detoxifies electrophilic chemicals
• Removal of glutamyl and glycinyl
• Addition of acetyl group to amino group to cysteinyl moiety
• Resulting in mercapturic acid
• Eliminated via urine

20. Other Reactions
• Acetylation
• Catalyzed by acetyltransferase
• Detoxifies or terminates drug activity
• Conjugation with amino acids like glycine, taurine

21. Other Reactions
• Methylation
• By methyltransferase; s-adenosylmethionine acts as a donor
• Most products are inactive
• Peroxidases: Prostaglandin H synthase, myeloperoxidase,
lactoperoxidase
• Prostaglandin H synthase has two catalytic activities:
• Cyclooxygensae (COX) converts arachidonic acid into cyclic endoperoxide-hydroperoxide
• Peroxidase that converts the hyroperoxidase into alcohol that can be oxidated
• COX-2 inhibitors could be apirin, ibuprofin

22. Phase III Reactions
• Includes drug transporters that influence ADME (absorption,
distribution, metabolism and excretion)
• Move drugs across barriers and target sites for accumulation
• Present in epithelial and endothelial liver cells, GI tract, kidney, blood-
brain barrier
• Conjugates and metabolites are excreted along with anionic groups
that act as affinity tags for many membrane transporters of MRP
family
• These proteins are of the ATP-binding cassette transporters family

23. References
• Graham, S., et al., How Similar are P450s and What Can Their Differences Teach
Us?, Archives of Biochemistry and Biophysics, Vol. 369, No. 1, September 1, pp.
24-29, 1999
• Remmel, R., et al., Concise Review of the Cytochrome P450s and Their Roles in
Toxicology, Toxicology Sciences 48, 151-156, 1999
• Abass., K., et al., Do Cytochrome P450 Enzymes Contribute to the Metabolism of
Xenobiotics in Human? http://cdn.intechopen.com/pdfs-wm/12397.pdf
• Homolya L., et al., (2003), "Multidrug resistance-associated proteins: Export
pumps for conjugates with glutathione, glucoronate or sulfate”, Biofactors 17 (1–
4): 103–14
• König J, et al., (1999), "Conjugate export pumps of the multidrug resistance
protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug
resistance". Biochim. Biophys. Acta. 1461 (2): 377 94