metabolism of xenobiotis, drugs, medicine, carcinogen generation by enzymes like cyt p450 mono oxigenases, prostaglandin synthase ect. alcohol metabolism, toxin metabolism, definition of genobiotics, biotransformation, detoxification. effects on health

1. Xenobiotics Metabolism
CYP450
Dr. Rajender Kumar
P.G. Biochemistry
Pt. B.D.S.P.G.I.M.S. Rohtak

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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)

13. 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

14. Location of Cytochrome P450

17. • 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

18. • 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

19. 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

21. Certain cytochrome P450s exist in polymorphic forms
(genetic isoforms), some of which exhibit low catalytic
activity
CYP2A6 involved in the metabolism of nicotine to conitine,
three alleles have been identified, a wild type and two null
or inactive alleles
Individuals with the null alleles, who have impaired
metabolism of nicotine, are apparently protected against
becoming tobacco- dependent smokers
because their blood and brain concentrations of nicotine
remain elevated longer
It has been speculated that inhibiting CYP2A6 may be a
novel way to help prevent and to treat smoking
Cytochrome P450

22. • 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

23. 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

24. 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+

25. Mechanism of CYP hydroxylation

26. PHASE I REACTIONS

27. • 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

28. Example of hyroxylation

30. 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
FMO
FAD
FMO
FADH2
NADP
+
FMO
FADHOOH
NADP+
FMO
FADHOH
NADP+
NADPH
+ H+
O2
X
XO
NADP+
H2O
FADHOOH is 4a-hydroperoxyflavin
FADHOH is 4a-hydroxyflavin
FMO
FAD

31. 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

32. 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

33. 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

34. 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
Non-Microsomal Oxidation Reactions

35. 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
isosteres
Enzymes: Non-microsomal hydrolases
however amide hydrolysis appears to be mediated by liver
microsomal amidases, esterases, deacylases
R1 C R2
O
δ−
δ+
O CH3
CO2H
O
OH
CO2
H
CH3
O
OH
ASA
Acetylsalicylic Acid
esterase

36. PHASE II REACTIONS
CONJUGATIONS

38. 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

39. Glucuronidation

41. 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

42. Glutathione (γ-glutamyl-cysteinylglycine) is a tripeptide
consisting of glutamic acid, cysteine, and glycine
It detoxify electrophilic chemicals
Conjugation with Glutathione

43. 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 moiety
The resulting compound is a mercapturic acid, a
conjugate of L acetylcysteine, which is then excreted in the
urine
Mechanism Conjugation with Glutathione

44. Reaction catalyzed by acetyltransferases present in the
cytosol of various tissues, particularly liver
Important for drugs with primary amino groups
The drug isoniazid, used in the treatment of tuberculosis,
is subject to acetylation
Acetylation does NOT increase water solubility
Causes Detoxification or termination of drug activity
Acetylation
isoniazid

45. 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

47. • with glycine, taurine
• xenobiotics with -COOH groups are the substrates
• endogenous example: conjugated bile acids
Approximately 76% of aspirin is metabolized through amino
acid conjugation
Salicyluric acid, the glycine conjugate of salicyclic acid, is
the main metabolite of aspirin
Conjugation with amino acids

48. Peroxidases
RH + O2
PHS
R-OOH + X or XH
PHS
MOx
LOx
ROH + XO or X
R-OOH + carcinogen
PHS
MOx
LOx
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

49. Prostaglandin H synthase
COOH
arachidonic acid
O2 + O2
O
O
COOH
OOH
PGG2
cyclooxygenase
peroxidase
X or 2XH
XO or 2X + H2O
O
O
COOH
OH
PGH2
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
PHS
NH
DNA
damage

54. Activation of prodrugs

55. CARCINOGENS

56. 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 deposits
A few PAHs are used to produce medicine, dyes, plastics, &
pesticides
Natural sources of PAHs include volcanoes and natural fires
Examples include Benzo(a)pyrene and Benzo(b)fluoranthene
PAH in environment

57. - 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 51
Industrial producers 20
Incineration & open burning 28
Vehicles 1
B(a)P in foodstuffs μg/kg
Charcoal broiled steak 8
Margarine 1-36
Sausages 4-50
Roasted coffee 1-13
Toast 0.5

58. PAHs effects
•Some PAHs have been shown to be cancer causing
•Chronic Bronchitis
•Skin Problems
•Allergies
Fetus 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

59. CYP/PHS
O
EH
HO
OH
CYP/PHS
HO
OH
O
benzo[a]pyrene (+) benzo[a]pyrene
7,8-oxide (-) benzo[a]pyrene
7,8-dihydrodiol
(+) benzo[a]pyrene
7,8-dihydrodiol-9,10-epoxide
ULTIMATE CARCINOGEN
HN
N
N
NO
HN
DNA
HO
OH
HO
BaP-N2
-dG DNA adduct
DNA
GST/GSH
OH
GS
inactive (excreted)
O
CYP/PHS
OH
OH
inactive
Phase II
Phase II and
excretion
PAHs metabolites

60. • 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

61. • 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

62. Aflatoxin
Aflatoxins 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

63. O
O
O
OO
OCH3
* *
isolated
e-
-rich double bond
aflatoxin
O
O
O
OO
OCH3
* *
O
ULTIMATE CARCINOGEN
CYP/PHS
DNA
NHN
N
N
O
NH2
DNA
O
O
O
OO
OCH3
HO
GST/GSH
O
O
O
OO
OCH3
* *GS
OH
EH
inactive (excreted)
O
O
O
OO
OCH3
* *HO
OH
* *
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

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

65. 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

66. Tobacco

67. • during cigarette burning?
• temperature about 900 o
C
• dried tobacco undergoes incomplete combustion
• nicotine partly passes to smoke, partly decomposes
• Nicotine: 0.9 mg/cig.
• Tar: 11 mg/cig.
Tobacco

68. 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

70. Toxic effects of xenobiotics
1. 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

71. 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

72. THANKS