Xenobiotics and cyt p450 by dr rajender

Xenobiotics MetabolismCYP450

Dr. Rajender KumarP.G. BiochemistryPt. B.D.S.P.G.I.M.S. Rohtak

Introduction

A xenobiotic is a foreign chemical substance found within an organism that is not normally, naturally produced by or expected to be present within that organism. It can also cover substances which are present in much higher concentrations than are usual

Examples- Drugs, food additives, pollutants, insecticides, chemical carcinogens etc

They enter body mainly with food or as medications Three principal entries: intestine, lungs, skin

Entry of xenobiotic into cells

• Simple diffusion – lipophilic substances, depends on concetration gradient (liver – freely permeable, big pores in cell membrane, most affected in poisoning)

• Facilitated diffusion – transporters• Active transport – primary, secondary• Endocytosis• xenobiotics structurally similar with physiological

substrates can utilize all available transport systems

Excretion of xenobiotics from cell

• primary active transport – needs energy: ATP hydrolysis

• special ATP-ases called ABC (ATP binding cassettes) localized in cell membranes, export xenobiotics from cells into ECF

• MRP (multidrug resistance proteins) – in increased expresion, they cause the resistance towards medicines

Excretion of xenobiotics from body

• chemically modified (more polar) xenobiotics are excreted by urine, bile, stool, or sweat

• volatile substance by lungs• excretion into human milk• intestinal deconjugation and reabsorption

sometimes occur - enterohepatic circulation

Biotransformation and detoxification

Biotransformation is Conversion of lipophilic to water-soluble chemicals catalyzed by enzymes in the liver and other tissues

In most cases, biotransformation lessens the toxicity of xenobiotics

The term “detoxification” is sometimes used for many of the reactions involved in the metabolism of xenobiotics

Increasingly humans are subjected to exposure to various foreign chemicals xenobiotics. Knowledge of the metabolism of xenobiotics is basic to a rational understanding of pharmacology and therapeutics pharmacy toxicology management of cancer, and drug addiction

All these areas involve administration of, or exposure to, xenobiotics

Biomedical importance

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 or converts inactive

drug to its active form

Purpose of Biotransformation

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

Metabolism of Xenobiotics

phase 1

1. Hydroxylation

Monooxygenases or cytochrome P450s

Hydroxylation may terminate the action of a drug

2. deamination, dehalogenation,

desulfuration, epoxidation,

peroxygenation, and reduction, hydrolysis

• Purpose– Introduction of polar functional groups in a molecules

♣ Increase a molecule’s polarity

♣ Does provide a site for phase II metabolism

Metabolism of Xenobiotics

Phase 2

conjugation with

glucuronic acid,

sulfate,

acetate,

glutathione,

methyl

or certain amino acids,

The overall purpose of phases II of metabolism of xenobiotics is to increase their water solubility (polarity) and thus excretion from the body

Xenobiotic-Metabolizing Enzymes (XME)

Phase 1• Cytochromes P450

• Flavin Containing Monooxygenase

• Epoxide Hydrolase

• Alcohol /Aldehyde Dehydrogenases

• Monoamine Oxidases

• Xanthine oxidase

Phase 2 “Transferases”Sulfotransferases (ST)UDP-glucuronosyltransferases (UGT)Glutathione S-transferases (GST)

Cytochrome P450

• superfamily of heme enzymes (many isoforms) can catalyze different reaction types, mainly hydroxylation

• Human:18 families, 43 subfamilies, 57 sequenced genes• can be induced and inhibited

• occur in most tissues (except of muscles and erythrocytes)

• the highest amount in the liver (ER)• exhibit genetic polymorphism (atypical biotransformations)• Nomencleature:

CYP1A2

family subfamily individual member of that subfamily

Location of Cytochrome P450

• They are present in highest amount in liver and small intestine but are probably present in all tissues

• In liver and most other tissues, they are present mainly in the membranes of the smooth endoplasmic reticulum

• In the adrenal, they are found in mitochondria as well as in the endoplasmic reticulum

• The various hydroxylases present in that organ play an important role in cholesterol and steroid biosynthesis

Cytochrome P450

• At least six isoforms of cytochrome P450 are

present in the endoplasmic reticulum of human liver,• acting on both xenobiotics and endogenous compounds• P450 metabolizes certain widely used solvents and also

components found in tobacco smoke, many of which are established carcinogens

• Most isoforms of cytochrome P450 are inducible

• Induction of cytochrome P450 has important clinical implications, since it is a biochemical mechanism of drug interaction

Cytochrome P450

Certain isoforms of cytochrome P450 (eg, CYP1A1) are particularly involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs) and related molecules

for this reason they were formerly called aromatic hydrocarbon hydroxylases (AHHs)

This enzyme is important in the metabolism of PAHs and in carcinogenesis produced by these agents

Cytochrome P450

Certain cytochrome P450s exist in polymorphic forms (genetic isoforms), some of which exhibit low catalytic activityCYP2A6 involved in the metabolism of nicotine to conitine, three alleles have been identified, a wild type and two null or inactive allelesIndividuals with the null alleles, who have impaired metabolism of nicotine, are apparently protected against becoming tobacco- dependent smokersbecause their blood and brain concentrations of nicotine remain elevated longerIt has been speculated that inhibiting CYP2A6 may be a novel way to help prevent and to treat smoking

Cytochrome P450

• some xenobiotics induce the synthesis of CYP – the metabolic capacity of CYP is enhanced

• if administered inducer + drug, both metabolized by the same CYP isoform and drug is metabolized faster, drug is less effective

• some xenobiotics inhibit CYP• the most common isoform CYP3A4 metabolizes more

than 120 different pharmaceutical drugs• inhibitors of CYP3A4 are e.g. macrolide antibiotics,

grapefruit (furanocoumarins), ketoconazole

• if administered inhibitor + drug, increased drug level, overdosing , side effects

Inducers and inhibitors of CYP450

cytochrome P450 contains three cofactors and two enzymes:

• NADPH+H+, FAD, heme

• NADPH:CYP reductase (separates 2 H 2 e- + 2H+) and

cytochrome P-450 hydroxylase

Lipids are also components of the cytochrome P450 system

The preferred lipid is phosphatidylcholine, which is the major lipid found in membranes of the endoplasmic reticulum

Components of cytochrome P450

Mechanism of CYP hydroxylation

The formation of hydroxyl group

monooxygenase: one O atom from O2 molecule is incorporated into substrate between C and H (R-H → R-OH )

The second O atom + 2H from NADPH+H+ give water

R-H + O2 + NADPH + H+ → R-OH + H2O + NADP+

2 e- + 2 H+

Mechanism of CYP hydroxylation

PHASE I REACTIONS

• Hydroxylation is the chief reaction involved

• The responsible enzymes are called monooxygenases and cytochrome P450s

• Hydroxylation by CYP450 occurs in endogenous and exogenous substrates

• Endoplasmic reticulum: squalene, cholesterol, bile acids, FA desaturation, prostaglandins, xenobiotics

• Mitochondria: steroidal hormones

hydroxylation

Example of hyroxylation

Flavin-containing Monooxygenase

•FAD-containing monooxygenases (FMO) oxidize nucleophilic nitrogen, sulfur and phosphorus heteroatoms of a variety of xenobiotics

• FMO’s are not inducible and are constitutively expressed

•Can be inhibited by other substrates

• Located in microsomal fraction of liver, kidney, and lung

FMOFAD

FMOFADH2NADP+

FMOFADHOOH

NADP+

FMOFADHOHNADP+

NADPH+ H+

O2

X

XO

NADP+

H2O

FADHOOH is 4a-hydroperoxyflavinFADHOH is 4a-hydroxyflavin

FMO

FAD

FMO Example Reactions

N

N

CH3 N

N

CH3

excretion

nicotine

O

N

O

CH3

H

N

O

CH3

OH

nicotine-1'-N-oxide

2-acetylaminofluorene (2-AAF)caricnogen

N-hydroxy-2-AAF

FMO

FMO

Epoxides are highly reactive and mutagenic or carcinogenic can form during Phase I (CYP/COX)

Epoxide hydrolase converting them into much less reactive dihydrodiols

There are 5 distinct forms of EH in mammals:

1. Microsomal epoxide hydrolase (mEH)

2. Soluble epoxide hydrolase (sEH)

3. Cholesterol epoxide hydrolase

4. LTA4 hydrolase

5. Hepoxilin hydrolase

mEH and sEH hydrolyze xenobiotic epoxides while the latter 3 hydrolases act on endogenous substrates

Epoxides

Hydrolysis in plasma by esterases (suxamethonium by cholinesterase)

Alcohol and aldehyde dehydrogenase in liver cytosolic (ethanol)

Monoamine oxidase in mitochondria (tyramine, noradrenaline, dopamine, amines)

Xanthine oxidase (6-mercaptopurine, uric acid production)

Enzymes for particular substrates (tyrosine hydroxylase, dopa-decarboxylase etc.)

Other (non-microsomal) Phase I reactions

RCH2NH2+O2+H2O2RCHO+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

Non-Microsomal Oxidation Reactions

Hydrolytic Reactions

R1 R2 Name Susceptibility to Hydrolysis

C O Ester Highest

C S Thioester

O O Carbonate

C N Amide

O N Carbamate

N N Ureide Lowest

Naming carbonyl - heteroatom groups

Hydrolyzes (adds water to) esters and amides and their isosteresEnzymes: Non-microsomal hydrolaseshowever amide hydrolysis appears to be mediated by liver microsomal amidases, esterases, deacylases

R1 C R2

Oδ−

δ+

O CH3

CO2H

O

OH

CO2H CH3

O

OH

ASA

Acetylsalicylic Acid

esterase

PHASE II REACTIONS

CONJUGATIONS

Glucuronidation

• 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

• Molecules such as 2-acetylaminofluorene (a carcinogen), aniline, benzoic acid, meprobamate (a tranquilizer), phenol, and many steroids are excreted as glucuronides

• The glucuronide may be attached to oxygen, nitrogen, or sulfur groups of the substrates

Glucuronidation

Some alcohols, arylamines, and phenols are sulfated

other biologic sulfation reactions are sulfation of steroids, glycosaminoglycans, glycolipids, and glycoproteins

The sulfate donor is adenosine 3-phosphate-5-phosphosulfate (PAPS)

Leads to inactive water-soluble metabolites

Sulfation

Glutathione (γ-glutamyl-cysteinylglycine) is a tripeptide consisting of glutamic acid, cysteine, and glycine

It detoxify electrophilic chemicals

Conjugation with Glutathione

The glutamyl and glycinyl groups belonging to glutathione are removed by specific Enzymes acetyl group (donated by acetyl- CoA) is added to the amino group of the remaining cysteinyl moietyThe resulting compound is a mercapturic acid, a conjugate of L acetylcysteine, which is then excreted in the urine

Mechanism Conjugation with Glutathione

Reaction catalyzed by acetyltransferases present in the cytosol of various tissues, particularly liverImportant for drugs with primary amino groupsThe drug isoniazid, used in the treatment of tuberculosis, is subject to acetylationAcetylation does NOT increase water solubilityCauses Detoxification or termination of drug activity

Acetylation

isoniazid

A few xenobiotics are subject to methylation by methyltransferases

S-adenosylmethionine is methyl donor

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

Methylation

• with glycine, taurine• xenobiotics with -COOH groups are the substrates• endogenous example: conjugated bile acidsApproximately 76% of aspirin is metabolized through amino

acid conjugationSalicyluric acid, the glycine conjugate of salicyclic acid, is

the main metabolite of aspirin

Conjugation with amino acids

Peroxidases

RH + O2

PHSR-OOH + X or XH

PHSMOxLOx

ROH + XO or X

R-OOH + carcinogen

PHSMOxLOx

active carcinogen(ie. aflatoxin)

1. Prostaglandin H synthase (PHS, COX1,2) (brain, lung, kidney, GI tract, urinary bladder)

2. Myeloperoxidase (MOx) (leukocytes)

3. Lactoperoxidase (LOx) (mammary gland)

Most oxidative biotransformations require reduced cofactors NADPH and NADH, except for peroxidases that couple the reduction of hydrogen peroxide and lipid hydroperoxides to the oxidation of other substrates called cooxidation

Prostaglandin H synthase

COOH

arachidonic acid

O2 + O2

O

O

COOH

OOH

PGG2

cyclooxygenase

peroxidaseX or 2XH

XO or 2X + H2O

O

O

COOH

OHPGH2

prostacyclinthromboxane A2prostaglandins(PGD2, PGE2)

PHS (COX) has two catalytic activities:

1. a cyclooxygenase (COX) that converts arachidonic acid to the cyclic endoperoxide-hydroperoxide PGG2)

2. a peroxidase (that converts the hydroperoxide to the corresponding alcohol PGH2) which can result in the oxidation of xenobiotics

3. COX-2 inhibitors include aspirin and ibuprofin

PHS can bioactivate carcinogens such as β-napthylamine, a bladder carcinogen

NH2 PHSNH

DNAdamage

Activation of prodrugs

CARCINOGENS

PAHs formed by:- incomplete combustion of organic matter such as coal,

wood, oil, petrol and diesel- coke production, vehicle and aircraft exhaust- smoking cigarettes- charbroiled meats

PAHs are also found in natural fuel depositsA few PAHs are used to produce medicine, dyes, plastics, &

pesticidesNatural sources of PAHs include volcanoes and natural fires

Examples include Benzo(a)pyrene and Benzo(b)fluoranthene

PAH in environment

- radicals formed by pyrolysis of hydrocarbons between

500 and 800ºC in zone of flame with insufficient O2

- C1 and C2 fragments combine in reducing atmosphere

to form condensed aromatics

- on cooling, PAHs condense onto existing particles –

their distribution reflects their differing thermodynamic

stability in O2 deficient flame

Mechanism of formation during combustion

Source % Heating, power production 51Industrial producers 20Incineration & open burning 28Vehicles 1

B(a)P in foodstuffs μg/kgCharcoal broiled steak 8Margarine 1-36Sausages 4-50Roasted coffee 1-13Toast 0.5

PAHs effects•Some PAHs have been shown to be cancer causing•Chronic Bronchitis•Skin Problems•AllergiesFetus is at greater risk and susceptibility :

•Growth retardation•Low birth weight•Small head circumference•Low IQ•Damage DNA•Disrupt endocrine systems, such as estrogen, thyroid, and steroids

CYP/PHS

O

EH

HO

OH

CYP/PHS

HO

OH

O

benzo[a]pyrene (+) benzo[a]pyrene7,8-oxide (-) benzo[a]pyrene

7,8-dihydrodiol

(+) benzo[a]pyrene7,8-dihydrodiol-9,10-epoxide

ULTIMATE CARCINOGEN

HNN

N

NO

HN

DNA

HO

OH

HO

BaP-N2-dG DNA adduct

DNA

GST/GSH

OHGS

inactive (excreted)

O

CYP/PHS

OH

OHinactive

Phase II

Phase II and excretion

PAHs metabolites

• The first reaction is an epoxidation. With benzo(a)pyrene, the product is the corresponding 7,8-epoxide that, in turn, is subject of epoxide hydrolases to form stereoisomeric dihydrodiols

• These are converted further to the 7,8-dihydrodiol-9,10-epoxide. The terminal oxidase is cytochrome P-450 (CYP1A1). The diol epoxide can exist in 4 stereoisomeric forms of which the key carcinogenic product is benzo(a)pyrene-r-7,t-8-diol-t-9,10-epoxide

• PAH epoxides can then be conjugated with GSH. This conjugation is regarded as a true detoxification reaction and is mediated by glutathione transferase (GSTM1)

PAHs metabolites

• The epoxides that are not conjugated with GSH are converted into phenols and diols. These PAH metabolites, however, are sometimes not sufficiently polar to be excreted and are therefore conjugated with glucuronic or sulfuric acids to enable excretion to occur

• In addition to conjugation, the hydroxylated derivatives of PAHs may undergo a number of oxidation and hydroxylation reactions. These include the conversion of phenols to phenol-epoxides and subsequently to diphenols and triols, diols to tetrols and diol-epoxides, and triols to triolepoxides and pentols

PAHs metabolites

AflatoxinAflatoxins are naturally occurring mycotoxins that are produced by many species of Aspergillus, a fungus.

They can be found on moldy peanuts, rice, corn and other crops.

Aflatoxin B1 is the most potent liver carcinogen.

Aspergillus fungus that procues aflatoxin Aspergillus fungus on corn

O

O

O

OO

OCH3

* *

isolated e--rich double bond

aflatoxin

O

O

O

OO

OCH3

* *O

ULTIMATE CARCINOGEN

CYP/PHS

DNA

NHN

N

NO

NH2

DNA

O

O

O

OO

OCH3

HO

GST/GSH

O

O

O

OO

OCH3

* *GSOH

EH

inactive (excreted)

O

O

O

OO

OCH3

* *HOOH

* *

AFB1 N7-DNA adduct

* electrophilic

some DNA activity

Epoxide hydrolase can detoxify aflatoxin-epoxide from binding to DNA, but still has some mutagenic activity

Aflatoxin metabolism

N-Hydroxylation of AAF

N-Hydroxylation of AAF is the first metabolic step towards the development of a carcinogenic agent

Further Metabolism of N-HydroxyAAF Produces Cancer

N-HydroxyAAF undergoes phase II metabolism to the ultimate carcingogen. The glucuronide pathway is also involved in carcinogenesis

Tobacco

• during cigarette burning?

• temperature about 900 oC

• dried tobacco undergoes incomplete combustion

• nicotine partly passes to smoke, partly decomposes

• Nicotine: 0.9 mg/cig.• Tar: 11 mg/cig.

Tobacco

Cigarette smoke contains• free base of nicotine – binds to receptors in the brain• CO – binds to hemoglobin to give carbonylhemoglobin

(tissue ischemia)

• nitrogen oxides – may generate reactive radical species

• polycyclic aromatic hydrocarbons (PAH)(pyrene, chrysene, benzo[a]pyrene …), main components of tar

• they can attack and damage DNA, carcinogens• other substances (N2, CO2, HCN, CH4, esters …)

Tobacco

Toxic effects of xenobiotics1. cell injury (cytotoxicity)

2. the reactive species of a xenobiotic may bind to a protein, altering its antigenicity. The xenobiotic is said to act as a hapten

3. reactions of activated species of chemical carcinogens with DNA are thought to be of great importance in chemical carcinogenesis

Factors that influence metabolism of xenobiotics

• 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

THANKS