Xenobiotics and cyt p450 by dr rajender
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Transcript of 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