INTRODUCTION:

Firstly CYP450 is discovered by R.T. Williams - in vivo, 1947. Brodie – in vitro, from late 40s till the 60s. Cytochrome P450 enzymes (hemoproteins) play an important role in the intra-cellular metabolism. It exists in prokaryotic and eukaryotic (plants insects fish and mammal, as well as microorganisms) Different P450 enzymes can be found in almost any tissue: liver, kidney, lungs and even brain. Plays important role in drugs metabolism and xenobiotics. Cytochrome P450 proteins in humans are drug metabolizing enzymes and enzymes that are used to make cholesterol, steroids and other important lipids such as prostacyclins and thromboxane-A2. These last two are metabolites of arachidonic acid⁵. Mutations in cytochrome P450 genes or deficiencies of the enzymes are responsible for several human diseases. Induction of some P450s is a risk factor in several cancers since these enzymes can convert procarcinogens to carcinogens. P450 enzymes play a major role in drug¹.

Fig.1. Structure of cytochrome P450 molecule complex to carbon monoxide

The name cytochrome P450 derives from the fact that these proteins have a heme group, and an unusual spectrum. The name cytochrome P450 is derived from the fact that these are colored ('chrome') cellular ('cyto') proteins, with a "pigment at 450 nm", so named for the characteristic Soret peak formed by absorbance of light at wavelengths near 450 nm when the heme iron is reduced (often with sodium dithionite) and complexed to carbon monoxide.Mammalian cytochrome P450s are membrane bound¹⁰. They were originally discovered in rat liver microsomes. Microsomes are turbid suspensions made by grinding up cells and isolating the membrane fraction that is still in suspension after the cell debris and mitochondria have been pelleted. These mixtures are very opaque to standard spectroscopy, because they scatter light so badly².The CO binds tightly to the ferrous heme, giving a difference between the absorbance of the two cuvettes. The spectrum was first observed in 1958.Other heme containing proteins don't absorb at 450 nm. The reason why Cytochrome P450 absorbs in this range is the unusual ligand to the heme iron. Four ligands are provided by nitrogen on the heme ring.Above and below the plane of the heme, there is room for two more ligands, the 5th and 6th ligands⁴. In cytochrome P450s, the 5th ligand is a thiolate anion, sulfur with a negative charge, S(-). The sulfur comes from a conserved cysteine at the heme binding region of the active site¹²

Fig.2. Cytochrome bind with ligand

Cytochrome P450 (abbreviated CYP, P450, infrequently and CYP450) is a very large and diverse superfamily of hemoproteins found in all domains of life^{[1]HYPERLINK
"http://en.wikipedia.org/wiki/Cytochrome_P450" \l
"cite_note-1#cite_note-1" [2]} .Cytochromes P450 use a plethora of both exogenous and endogenous compounds as substrates in enzymatic reactions. Usually they form part of multicomponent electron transfer chains, called P450-containing systems.The most common reaction catalysed by cytochrome P450 is a monooxygenase reaction, e.g. insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water:

RH + O₂ + 2H⁺ + 2e^– → ROH + H₂O:

CYP enzymes have been identified from all lineages of life, including mammals, birds, fish, insects, worms, sea squirts, sea urchins, plants, fungi, slime molds, bacteria and archaea. More than 8100 distinct CYP sequences are known.³

Mechanism

Fig.3.Catalytic cycle of CYP 450

1: The substrate binds to the active site of the enzyme, in close proximity to the heme group, on the side opposite to the peptide chain. The bound substrate induces a change in the conformation of the active site, often displacing a water molecule from the distal axial coordination position of the heme iron^6.

2: The change in the electronic state of the active site favors the transfer of an electron from NAD(P)H via cytochrome P450 reductase or another associated reductase^{HYPERLINK
"http://en.wikipedia.org/wiki/Cytochrome_P450" \l "cite_note-P450pot-8#cite_note-P450pot-8"
9}.

3: Molecular oxygen binds covalently to the distal axial coordination position of the heme iron. The cysteine ligand is a better electron donor than histidine, with the oxygen consequently being activated to a greater extent than in other heme proteins

4: A second electron is transferred via the electron-transport system, either from cytochrome P450 reductase, ferredoxins, or cytochrome b5, reducing the dioxygen adduct to a negatively charged peroxo group. This is a short-lived intermediate state.

5: The peroxo group formed in step 4 is rapidly protonated twice by local transfer from water or from surrounding amino-acid side chains, releasing one water molecule, and forming a highly reactive iron(V)-oxo species^8.

6: After the product has been released from the active site, the enzyme returns to its original state, with a water molecule returning to occupy the distal coordination position of the iron nucleus.

Sulfur: An alternative route for mono-oxygenation is via the "peroxide shunt": interaction with single-oxygen donors such as peroxides and hypochlorites can lead directly to the formation of the iron-oxo intermediate, allowing the catalytic cycle to be completed without going through steps 3, 4 and 5.⁴ A hypothetical peroxide "XOOH" is shown in the diagram.

Carbon: If carbon monoxide (CO) binds to reduced P450, the catalytic cycle is interrupted. This reaction yields the classic CO difference spectrum with a maximum at 450 nm.

Table.1. CYP450 Inducers, Substrates, and Inhibitors

P-450 isoform	Substrate	Inducer	Inhibitor
CYP1A2	Amitriptyline Imipramine	Omeprazole Cigarette smoke	Fluvoxamine Ciprofloxacin
CYP2A6	Halothane	Phenytoin	Tranylcypromine
CYP3C9	Diazepam Diclofenac S-warfarin	Barbiturates Carbamazepin Primidone
CYP2C19	Omeprazol		Tranylcypromine
CYP2D6	Codeine Dihydrocodeine Propranolol		Ritonavir SSRIs(Selective Serotonin reuptake inhibitors)
CYP2E1	Enflurane Halothane	Alcohol(chronic) Isoniazid	Cimetidine Disulfiram
CYP3A4	Amiodarone Terfenadine	Carbamazebine Phyneoin	Erythromycin Traconazole
CYP4A1	Testosterone	Clofibrate

Humans CYP450 -18 families, 43 subfamilies:

n CYP1 drug metabolism (3 subfamilies, 3 genes, 1 pseudo-gene)

n CYP2 drug and steroid metabolism (13 subfamilies, 16 genes, 16 pseudo-genes)

n CYP3 drug metabolism (1 subfamily, 4 genes, 2 pseudo-genes)

n CYP4 arachidonic acid or fatty acid metabolism (5 subfamilies, 11 genes, 10Pseudo-genes)

n CYP5 Thromboxane A2 synthase (1 subfamily, 1 gene)CYP7A bile acid biosynthesis 7-alpha hydroxylase of steroid nucleus (1subfamily member)

n CYP7B brain specific form of 7-alpha hydroxylase (1 subfamily member)

n CYP8A prostacyclin synthase (1 subfamily member)

n CYP8B bile acid biosynthesis (1 subfamily member)

n CYP11 steroid biosynthesis (2 subfamilies, 3 genes)

n CYP17 steroid biosynthesis (1 subfamily, 1 gene) 17-alpha hydroxylase

n CYP19 steroid biosynthesis (1 subfamily, 1 gene) aromatase forms estrogen

n CYP20 Unknown function (1 subfamily, 1 gene)

n CYP21 steroid biosynthesis (1 subfamily, 1 gene, 1 pseudo-gene)

n CYP24 vitamin D degradation (1 subfamily, 1 gene)

n CYP26A retinoic acid hydroxylase important in development (1 subfamily member)

n CYP26B probable retinoic acid hydroxylase (1 subfamily member)

n CYP26C probable retinoic acid hydroxylase (1 subfamily member)

n CYP27A bile acid biosynthesis (1 subfamily member)

n CYP27B Vitamin D3 1-alpha hydroxylase activates vitamin D3 (1 subfamily member)

n CYP27C Unknown function (1 subfamily member)

n CYP39 unknown function (1 subfamily member)

n CYP46 cholesterol 24-hydroxylase (1 subfamily member)

n CYP51 cholesterol biosynthesis (1 subfamily, 1 gene, 3 pseudogenes) lanosterol 14-alpha demethylase

Fig.4. Hydroxylation reaction

P450s catalyze: Types of reactions:

the most important is hydroxylation. These enzymes are called mixed function oxidases or monooxygenases, because they incorporate one atom of molecular oxygen into the substrate and one atom into water. They differ from dioxygenases that incorporate both atoms of molecular oxygen into the substrate^12,14.

Foreign chemicals or drugs are also called xenobiotics. Cytochrome P450s play an important role in xenobiotic metabolism, especially for lipophilic drugs. The metabolism of these compounds takes place in two phases. Phase I is chemical modification to add a functional group that can be used to attach a conjugate¹⁵. The conjugate makes the modified compound more water soluble so it can be excreted in the urine. Many P450s add a hydroxyl group in a Phase I step of drug metabolism. The hydroxyl then serves as the site for further modifications in Phase 2 drug metabolism^15,16.

Fig.5. Xenobiotic reaction

Two electrons are acquired from NADPH and migrate from FAD to FMN, then to the P450 heme iron:

NADPH + H⁺ + O₂ + RH NADP⁺ + H₂O + R-OH

CYP families in humans:

Humans have 57 genes and more than 59 pseudo genes divided among 18 families of cytochrome P450 genes and 43 subfamilies¹⁸.

P450s in animals

Many animals have as many or more CYP genes than humans do. For example, mice have genes for 101 CYPs, and sea urchins have even more (perhaps as many as 120 genes)^[19] .Most CYP enzymes are presumed to have monooxygenase activity, as is the case for most mammalian CYPs that have been investigated (except for e.g. CYP19 and CYP5).CYPs have been extensively examined in mice, rats, and dogs, and less so in zebrafish, in order to facilitate use of these model organisms in drug discovery and toxicology.CYPs have also been heavily studied in insects, often to understand pesticide resistance.

P450s in bacteria:

Bacterial cytochromes P450 are often soluble enzymes and are involved in critical metabolic processes. There are examples that have contributed significantly to structural and mechanistic studies are listed here, but many different families exist^20.

Table:2. CYP450 families I n human

Family	Function	Members	Names
CYP1	drug and steroid (especially estrogen) metabolism	3 subfamilies, 3 genes, 1 pseudo gene	CYP1A1, CYP1A2, CYP1B1
CYP2	drug and steroid metabolism	13 subfamilies, 16 genes, 16 pseudo genes	CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1
CYP3	drug and steroid (including testosterone) metabolism	1 subfamily, 4 genes, 2 pseudo genes	CYP3A4, CYP3A5, CYP3A7, CYP3A43
CYP4	arachidonic acid or fatty acid metabolism	6 subfamilies, 11 genes, 10 pseudogenes	CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, CYP4Z1
CYP5	thromboxane A₂ synthase	1 subfamily, 1 gene	CYP5A1
CYP7	bile acid biosynthesis 7-alpha hydroxylase of steroid nucleus	2 subfamilies, 2 genes	CYP7A1, CYP7B1
CYP8	varied	2 subfamilies, 2 genes	CYP8A1 (prostacyclin synthase), CYP8B1 (bile acid biosynthesis)
CYP11	steroid biosynthesis	2 subfamilies, 3 genes	CYP11A1, CYP11B1, CYP11B2
CYP17	steroid biosynthesis, 17-alpha hydroxylase	1 subfamily, 1 gene	CYP17A1
CYP19	steroid biosynthesis: aromatase synthesizes estrogen	1 subfamily, 1 gene	CYP19A1
CYP20	unknown function	1 subfamily, 1 gene	CYP20A1
CYP21	steroid biosynthesis	2 subfamilies, 2 genes, 1 pseudo gene	CYP21A2
CYP24	vitamin D degradation	1 subfamily, 1 gene	CYP24A1
CYP26	retinoic acid hydroxylase	3 subfamilies, 3 genes	CYP26A1, CYP26B1, CYP26C1
CYP27	varied	3 subfamilies, 3 genes	CYP27A1 (bile acid biosynthesis), CYP27B1 (vitamin D3 1-alpha hydroxylase, activates vitamin D3), CYP27C1 (unknown function)
CYP39	7-alpha hydroxylation of 24-hydroxycholesterol	1 subfamily, 1 gene	CYP39A1
CYP46	cholesterol 24-hydroxylase	1 subfamily, 1 gene	CYP46A1
CYP51	cholesterol biosynthesis	1 subfamily, 1 gene, 3 pseudogenes	CYP51A1 (lanosterol 14-alpha demethylase)

 Cytochrome P450cam (CYP101) originally from Pseudomonas putida has been used as a model for many cytochromes P450 and was the first cytochrome P450 three-dimensional protein structure solved by x-ray crystallography.

 Cytochrome P450 eryF (CYP107A1) originally from the actinomycete bacterium Saccharopolyspora erythraea is responsible for the biosynthesis of the antibiotic erythromycin by C6-hydroxylation of the macrolide 6-deoxyerythronolide B.

 Cytochrome P450 BM3 (CYP102A1) from the soil bacterium Bacillus megaterium catalyzes the NADPH-dependent hydroxylation of several long-chain fatty acids at the ω–1 through ω–3 positions

P450s in fungi:

The commonly used imidazole and triazole-class antifungal drugs work by inhibition of the fungal cytochrome P450 14α-demethylase. This interrupts the conversion of lanosterol to ergosterol, a component of the fungal cell membrane²¹.

P450s in plants:

Plant cytochromes P450 are involved in a wide range of biosynthetic reactions, leading to various fatty acid conjugates, plant hormones, defensive compounds, or medically important drugs. Terpenoids, which represent the largest class of characterized natural plant compounds, are often substrates for plant CYPs differences in humans, mice and rats.

Not all mammals have the same exact sets of P450 enzymes. Humans have 4 CYP2csWhile mice have 15. This has implications for drug testing in animals. One has to be concerned that studying the effect of a drug in mice or rats may not be relevant to humans, since the drug metabolizing systems are different. Beagle dogs are sometimes used in drug experiments, because their drug metabolism is supposed to be closer to humans than rodents^[5][3] .

Use of a P450 for gene therapy in cancer

Fig.6. p450 in gene therapy

CYP1A2 can activate procarcinogens to carcinogens. The induction of this enzyme may be a cancer risk. The activation of a prodrug to an active form by a P450 mediated reaction has been exploited to fight cancer. A vector with a P450 gene on it (and a P450 reductase gene) can be injected into cancer tumors. Some of these cells take up the vector and express The P450 and its reductase.Then a non-toxic prodrug is administered that is converted by the P450 into a toxic compound toxin gets shared around and the tumor dies²³.

Clinically important aspects of CYP450 drug metabolism:

1. Genetic polymorphism

2. Drug- Drug interaction

A) Enzyme induction B) Enzyme Inhibition

Genetic polymorphism

Drug-Drug Interaction:

Enzyme inhibition

Practical application of CYP450 is used in Roche AmpliChip Cytochrome P450 Genotyping test and Affymetrix GeneChip Microarray Instrumentation System - K042259

The Roche AmpliChip Cytochrome P450 Genotyping test for use on the Affymetrix GeneChip Microarray Instrumentation System is new laboratory test systems that will help doctors personalize treatment options for their patients. Doctors can use a patient’s genetic information to help them determine appropriate drugs and doses to prescribe. This will help minimize harmful drug reactions and prevent patients from being improperly treated with sub-optimal doses. This system uses DNA extracted from a patient’s blood to detect certain common genetic mutations that alter the body’s ability to break down (metabolize) specific types of drugs. The enzyme produced from the gene that is tested, called cytochrome P4502D6 (CYP4502D6), is active in metabolizing many types of drugs including antidepressants, antipsychotics, beta-blockers, and some chemotherapy drugs¹⁷. Variations in this gene can cause a patient to metabolize these drugs abnormally fast, abnormally slow, or not at all. For example, the same dose that is safe for a patient with one variation might be too high (and therefore toxic) to a patient with a different variation who cannot metabolize the drug. With genetic information for this gene, many harmful reactions resulting from inappropriate dosing and treatment may be significantly reduced as clinicians can adjust the patient’s regimen accordingly.

How does it work? A doctor orders the genetic test in patients to gather information on the predicted metabolic activity of their enzyme encoded by CYP4502D6.

 A sample of blood is collected and taken to the lab.

 The lab extracts DNA from the blood sample.

 The lab processes and applies the DNA to the Cytochrome P450 Genotyping test.

 The GeneChip Microarray Instrumentation System reads the test.

 The genetic result for the Cytochrome P450 Genotyping test is sent to the doctor.

 The doctor uses the Cytochrome P450 genetic test results, clinical evaluation and other lab tests as an aid in individualizing patient treatment options.

The AmpliChip Cytochrome P450 Genotyping System is used to help a clinician determine if a patient has mutations in their CYP4502D6 gene that may affect their ability to metabolize certain drugs. The test system is designed to aid the doctor in making individualized treatment decisions. The AmpliChip Cytochrome P450 Genotyping System may help the doctor determine if a patient is at risk of adverse drug reactions or sub-optimal drug response. When should it not be used. As the only test to determine specific drug dose. Other clinical information and patient history should primarily be considered.

 To aid in predicting a patient’s drug response for drugs that are not metabolized by the enzyme encoded by Cytochrome P4502D6.

 To aid in predicting a patient’s response to drugs for which the mutant Cytochrome P4502D6 phenotype has not been clearly establish.

REFERENCES:

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2. International Union of Pure and Applied Chemistry. "cytochrome P450". Compendium of Chemical Terminology Internet edition. Danielson P (2002). "The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans". Curr Drug Metab 3(6): 561–97.

3. Meunier B, de Visser SP, Shaik S (September 2004). "Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes". Chem. Rev. 104 (9): 3947–80.

4. Poulos TL, Finzel BC, Howard AJ (June 1987). "High-resolution crystal structure of cytochrome P450cam". J. Mol. Biol. 195 (3): 687–700. ^a ^b Ortiz de Montellano, Paul R.; Paul R. Ortiz de Montellano (2005). Cytochrome P450: structure, mechanism, and biochemistry (3rd ed.). New York: Kluwer Academic/Plenum Publishers. Sligar SG, Cinti DL, Gibson GG, Schenkman JB (October 1979). "Spin state control of the hepatic cytochrome P450 redox potential". Biochem. Biophys. Res. Commun. 90 (3): 925–32.

5. Guengerich FP (January 2008). "Cytochrome p450 and chemical toxicology". Chem. Res. Toxicol. 21 (1): 70–83.

6. Bailey DG, Dresser GK (2004). "Interactions between grapefruit juice and cardiovascular drugs". Am J Cardiovasc Drugs 4 (5): 281–97. Harder DR, Campbell WB, Roman RJ. (1995) Role of cytochrome P-450 enzymes and metabolites of arachidonic acid in the control of vascular tone. J Vasc Res; 32: 79–92.

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9. McGiff JC, Quilley J. (1999). 20-HETE and the kidney: resolution of old problems and new beginnings. Am J Physiol (Regulatory Integrative Comp Physiol); 277: R607–R623.

10. Omata K, Abraham NG, Escalante B, Schwartzman ML. (1992). Age-related changes in renal cytochrome P-450 AA metabolism in SHRs. Am J Physiol (Renal Physiol); 262: F8–F16.

11. Omata K, Abraham NG, Schwartzman ML. (1992). Renal cytochrome P-450-arachidonic acid metabolism: localization and hormonal regulation in SHR. Am J Physiol (Renal Physiol); 262: F591–F599.

12. Madhun ZT, Goldthwait DA, McKay D, Hopfer U, Douglas JG. (1991). An epoxygenase metabolite of arachidonic acid mediates angiotensin II-induced rises in cytosolic calcium in rabbit proximal tubule epithelial cells. J Clin Invest; 88: 456–461.

13. Zeratsky K (2008-11-06). "Grapefruit juice: Can it cause drug interactions?" Ask a food & nutrition specialist. MayoClinic.com. http://www.mayoclinic.com/health/food-and-nutrition/AN00413.

14. Chaudhary A, Willett KL (January 2006). "Inhibition of human cytochrome CYP 1 enzymes by flavonoids of St. John's wort". Toxicology 217 (2-3): 194–205.

15. Strandell J, Neil A, Carlin G (February 2004). "An approach to the in vitro evaluation of potential for cytochrome P450 enzyme inhibition from herbals and other natural remedies". Phytomedicine 11 (2-3): 98–104. Kroon LA (September 2007). "Drug interactions with smoking". Am J Health Syst Pharm 64 (18): 1917–21. Zhang JW, Liu Y, Cheng J, Li W, Ma H, Liu HT, Sun J, Wang LM, He YQ, Wang Y, Wang ZT, Yang L (2007). "Inhibition of human liver cytochrome P450 by star fruit juice". J Pharm Pharm Sci 10 (4): 496–503.

16. Goldstone JV, Hamdoun A, Cole BJ, Howard-Ashby M, Nebert DW, Scally M, Dean M, Epel D, Hahn ME, Stegeman JJ (December 2006). "The chemical defensome: environmental sensing and response genes in the Strongylocentrotus purpuratus genome". Dev. Biol. 300 (1): 366–84.

17. Narhi L, Fulco A (06/05/1986). "Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium". J Biol Chem 261(16): Girvan H, Waltham T, Neeli R, Collins H, McLean K, Scrutton N, Leys D, Munro A (2006). "Flavocytochrome P450 BM3 and the origin of CYP102 fusion species". Biochem Soc Trans 34 (Pt 6): 1173–7.

18. Omata K, Abraham NG, Schwartzman ML. (1992). Renal cytochrome P-450-arachidonic acid metabolism: localization and hormonal regulation in SHR. Am J Physiol (Renal Physiol); 262: F591–F599.

19. Madhun ZT, Goldthwait DA, McKay D, Hopfer U, Douglas JG. (1991). An epoxygenase metabolite of arachidonic acid mediates angiotensin II-induced rises in cytosolic calcium in rabbit proximal tubule epithelial cells. J Clin Invest; 88: 456–461.

20. Zeratsky K (2008-11-06). "Grapefruit juice: Can it cause drug interactions?". Ask a food & nutrition specialist. MayoClinic.com.

21. Chaudhary A, Willett KL (January 2006). "Inhibition of human cytochrome CYP 1 enzymes by flavonoids of St. John's wort". Toxicology 217 (2-3): 194–205.

22. Strand ell J, Neil A, Carlin G (February 2004). "An approach to the in vitro evaluation of potential for cytochrome P450 enzyme inhibition from herbals and other natural remedies". Phytomedicine 11 (2-3): 98–104.20.

23. Kroon LA (September 2007). "Drug interactions with smoking". Am J Health Syst Pharm 64 (18): 1917–21.

Received on 24.12.2009 Modified on 09.02.2010

Asian J. Research Chem. 3(2): April- June 2010; Page 243-248