BIOTRANSORMATION OF

XENOBIOTICS

Other Xenobiotic
metabolising
Enzymes and Drug
Bioactivation

Prof. S. Mukanganyama
Department of Biochemistry
MBCHB & BDS 1 2019 L6

1
 OUTLINE
2

• Xenobiotic metabolism pathways

• Cytochrome P450 Enzymology
• Nucleophilic reaction trapping enzymes
• Epoxide hydrolase, Glutathione transferases
• Conjugation enzymes
• UDPGTase, Sulphotransferases, N-acetyltransferases
• Drug Transporters- ABC transporters
• Factors affecting the activity of drug metabolism
enzymes-Pharmacogenetics, Inhibition, Induction
• Drug Bioactivation and link to toxicity
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 Bioactivation (Toxication)
4

 Formation of harmful or highly reactive

metabolic from relatively inert/nontoxic
chemical compounds is called bioactivation or
toxication.
 Bioactive metabolites interact with the body
tissues to precipitate one or more forms of
toxicities such as:
 carcinogenesis,

 teratogenesis,

 tissue necrosis.
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 Bioactivation (Toxication)
6

 Reactions generally catalyzed by cytochrome

P450-dependent monooxygenase systems,
 Some other enzymes like those in intestinal flora
are also involved in some cases.
 Reactive metabolites primarily belong to three
main categories—
 electrophiles, free radicals, and nucleophiles
 Formation of electrophiles and free radicals from
relatively harmless substances/xenobiotics
account for most toxicities
 Thalidomide
7

1. The “Wonder Drug” Gone

Wrong

2. The drug that caused many

heartaches
 Drug Development
8

•Stolberg, Germany 1954

•Chemie Grünenthal
–Desired low-cost method production of antibiotics
made from peptides
–Patented α-phthalimidoglutarimide (thalidomide)
•Unable to demonstrate antibiotic activity or
any medicinal or sedative effects in mice or rats
•Extremely high doses were not fatal nor were
other side effects detected in animal testing
•Therefore, thalidomide described as “nontoxic”
 Manipulation of Thalidomide
9
Test Results
• Early 1955, exploration of possibility of human
sedative through distribution of free samples
• First prescribed for epileptic seizures
• Patients reported “calming and sleep-inducing
effects”
• Germany required confirmation of the purported
effect on animals
• Grünenthal researchers invented “jiggle cage”
• Explicit claim of safety lead to sale even
sometimes as an OTC
 Thalidomide
10

•A teratogen-Causes of foetal disabilities

•Targets blood vessels.

•Blocks growth and so tends to target parts of the body

undergoing growth.

•Pregnant women took the drug to combat morning sickness,

but damage was being done to the rapidly growing foetus.

•Many of the thalidomide babies experienced phocomelia

because morning sickness can appear around the time of foetal
limb growth.
 The Damage
11

• 10,000-12,000 thalidomide babies

• 46 affected countries

• Drawn-out legal battles

• Disrupted families
 Negative Family Dynamic
12

• Divorce
• Abandonment
• Suicide (rare, but occurred)
• Sibling Resentment
• Infanticide (Belgium case)
 THALIDOMIDE TIMELINE
13

•1953 - Drug created in Germany

•1957 - Marketed to the public as a 'wonder drug' for insomnia,
colds, coughs and headaches
•1958 - Licensed in the UK
•1961 - William McBride, an Australian doctor , wrote to the
Lancet after noticing a sudden increase in the number of
deformed babies born at his hospital - all to mothers who had
taken thalidomide.
The drug was withdrawn from use in November that year.
•1968 - First UK compensation settlements reached with
manufacturers Distillers Biochemicals Limited
•1998 - Approved by the US's Food and Drug Administration as a
treatment for a complication of leprosy
•2004 - Thalidomide available on a named patient basis and as
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Thalidomide binds with a protein called cereblon

(CRBN) which helps trigger an enzyme (E3 ubiquitin
ligase) responsible for limb formation
 Potent human teratogen,
limb. Malformations after
martenal usage

16
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Only around half of the affected babies survived - 455 of them in the UK.
Distillers Biochemicals Limited, marketed the drug in the UK.
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 Thalidomide Today
20

• “What was once the most feared drug in

pharmaceutical armory could become
one of its most valuable.”

• Researchers look at the interactions

that caused deformities to discover the
safety of the drug in other uses
 Current Uses
21

• Cancer treatment
– Inhibit tumors directly
• Drug will stop blood vessels from forming in and around
tumors
– Activate immune system
– Anti-inflammatory
• Promising results seen in most intractable
cancers
 Other Promising Uses
22

• Standard Treatment for leprosy (ENL)

– Drug is FDA approved for ENL
– Changes body’s immunological and
inflammatory response to bacteria
– Used to heal lesions and skin ulcerations

• Currently used in some HIV patients

– Heal ulcers
– Diminish lean body mass loss during
wasting stages
 Precautions and Concerns
23

• Required pregnancy test in most cases

• Contraceptives must be accessible
• Education of the drug required
• The drug has potential to stay in body
for years
• Drug may enter semen and men could
affect female partners
 COOH
Prostaglandin H synthase
arachidonic acid PHS (COX) has two catalytic activities:
O2 + O2 1. a cyclooxygenase (COX) that converts
24
cyclooxygenase
arachidonic acid to the cyclic endoperoxide-
COOH hydroperoxide PGG2)

O 2. a peroxidase (that converts the

hydroperoxide to the corresponding alcohol
O PGH2) which can result in the oxidation of
OOH xenobiotics.
PGG2 3. COX-2 inhibitors include aspirin and
X or 2XH
peroxidase ibuprofin
PHS can bioactivate carcinogens such
XO or 2X + H2 O as β-napthylamine, a bladder
COOH
carcinogen.
O
NH2 NH
O PHS DNA
damage
OH
PGH 2

pr osta gla nd ins thro mbo xa ne A 2 pro sta cyclin

(P GD 2, PG E2)
Benzene: targets liver, kidney, lung, heart, and brain and can cause DNA strand breaks,
chromosomal damage, protein binding—can cause bone marrow suppression and
leukemia
• Exposure can arise from vapors from glues, paints, furniture wax, detergents (also
25 now limited)
• Air around hazardous waste sites or gas stations, exhaust from cars, industrial
emissions
 Flavin-containing
Monooxygenase
26 •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.
 Catalytic cycle of FMO

+ FMO
NADP FMO
FAD NADPH
27
H2O FAD + H+

FMO FMO
FADHOH+ FADH 2
NADP NADP
+

XO O2

FMO
FADHOOH
X NADP+

FAD HOOH is 4a-hydroperoxyflavin

FAD HOH is 4a-hydroxyflavin
 FMO Example Reactions
28
FMO
excretion
N N
O
N CH3 CH3
N
nicotine nicotine-1'-N-oxide

O FMO O

N CH3 N CH3
OH
H
2-acetylaminofluorene (2-AAF) N-hydroxy-2-AAF
caricnogen
 FMO-catalyzed bioactivation
O O
S O
29 S S
FMO C FMO
C NH2 C
NH2 NH2

thiobenzamide thiobenzamide S-oxide thiobenzamide S,S-dioxide

carcinogen (sulfine) (sulfene)

covalent binding

O
S
O C
C NH2
NH2

benzamide
oxathiirane
intermediate
 Benzo[a]pyrene
30

 Percival Pott in 1775 recognized the role of

soot in scrotal cancer among chimney sweeps
 Problem was solved by instructing chimney
sweeps to clean themselves after work.
 Benzo[a]pyrene
31

 The causative agents were polycyclic aromatic

hydrocarbons and a carcinogen culprit,
benzo[a]pyrene (BaP), was isolated from coal
tar in 1933.
 BaP is found in charbroiled meats, tobacco
smoke, coal tar.
 BaP is a potent carcinogen upon bioactivation.
 CYP/PHS O
CYP/PHS EH

HO HO
O OH OH
benzo[a]pyrene (+) benzo[a]pyrene
32 (+) benzo[a]pyrene
7,8-oxide (-) benzo[a]pyrene
7,8-dihydrodiol-9,10-epoxide
7,8-dihydrodiol
ULTIMATE CARCINOGEN
CYP/PHS GST/GSH

DNA
Phase II and
excretion
O N
GS HN N
O OH N DNA
inactive (excreted) HN
HO

HO
OH

OH BaP-N2-dG DNA adduct

OH
inactive

Phase II
 Benzopyrene Reacting with
33
Guanine in DNA
 Aflatoxin
34

Naturally occurring mycotoxins that are produced by many

species of Aspergillus, a fungus.
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 * electrophilic O O
isolated O
e--rich double bond
O
O * *
O * *
CYP/PHS

35 O O

O O OCH3
OCH3
aflatoxin ULTIMATE CARCINOGEN

EH GST/GSH
DNA
O O O O
OH OH NH2
HO O * * GS O * * N NH
DNA O O
N
O O HO O
N O * *
O OCH3 O OCH3
O
some DNA activity inactive (excreted) O OCH3
AFB1 N7-DNA
adduct

Epoxide hydrolase can detoxify aflatoxin-epoxide from binding to DNA, but still has
some mutagenic activity
 CANCER AND APOPTOSIS
36
 Basic knowledge of cancer
37

• What is Cancer?
• How does cancer occur?
• How many types of cancers?
• Current therapeutic strategies for
cancers
 Cancer
38

 Neoplasia (Cancer): a new growth

 an abnormal mass of tissue whose growth
exceeds and is uncoordinated with that of
normal tissues.
 Loss of responsiveness to normal growth
control.
 Cancer = Tumour = Neoplasm - study -
Oncology
 Malignant cells proliferate to produce progeny
that are also malignant.
 Cancer
39

 Malignancy are encoded in the genes of the

cancer cell i.e. is a genetic disease
 Cells derived clonally from a single precursor
cell
 Transformed cells
 Replicate without control
 "Parasites" -compete with normal tissues for
metabolic needs e.g. flourish in patients who
are otherwise wasting
 Partial autonomy
 Cellular Basis of Cancer
40

 A collection of diseases
characterized by abnormal and
uncontrolled growth

 Arises from a loss of normal growth

control

 In normal tissues, the rates of new

cell growth and old cell death are
kept in balance

 In cancer, this balance is disrupted

 This disruption can result from

1) uncontrolled cell growth or
2) loss of a cell's ability to undergo 40
apoptosis
 CANCER AS A CELLULAR
DISEASE
41

 Latent period -10-20 years.

 Unlimited proliferation
 Contain products of mutated oncogenes
 Expression of foetal genes.
 Evade the immune response
 Rapidly proliferating cells more sensitive to
chemotherapeutic agents
 Resistant to multiple anticancer drugs:
 Ability to metastasise- ability to form distant new
growths
 BENIGN TUMOURS
42

 Localised and cannot spread

 Amenable to surgical removal

 Well differentiated cells, resemble very closely

their normal counterparts

 Develop an enclosing fibrous capsules that

separate them from host tissue
43

Expansile Growth pattern

 Malignant Tumours
44

 True " cancers" –crab-Adhere to any part they

seize upon.
 Invade and destroy structures, spread to distant
sites (metastasise) to cause death.
 Nuclear -cytoplasmic ratio 1:1 instead of the
normal 1:4 or 1:6.
 Giant cells with either one enormous nucleus or
several nuclei.
 Nuclei variable + bizarre in size and shape
 No recognisable patterns of orientation to each
other.
45

Invasive growth pattern

  Malignant tumors can invade other tissues and
may kill the organism
46

Lymph
vessels

Tumor

Glandular
tissue

Metastasis

1 A tumor grows 2 Cancer cells invade 3 Cancer cells spread

from a single neighboring tissue. through lymph and
cancer cell. blood vessels to other
parts of the body.
 Neoplasia
47

 Neoplasia is a new growth of tissue

- a tumor
 Malignant neoplasias are true
cancers
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Cancer Cell Do Not Grow Faster

Than
Normal Cells
Rather, Their Growth is Just
Uncontrolled
 Cancer: disruption of cellular
51
equilibrium

Proliferation Differentiation Death

 Invasion and Metastasis
52

 Abnormal cells
proliferate and spread
(metastasize) to other
parts of the body

 Invasion - direct
migration and
penetration into
neighboring tissues

 Metastasis - cancer
cells penetrate into
lymphatic system and
blood vessels
 Three types of Malignant Cells
53

• Carcinomas; 90% of cancers, - cancers of

epithelial cells
• Sarcomas; rare and consist of tumors of
connective tissues (connective tissue,
muscle, bone etc.)
• Leukemias and lymphomas; constitute 8%
of tumors. “ liquid tumors”.
• Leukemias arise from blood forming cells
• Lymphomas arise from cells of the
immune system (T and B cells).
 What causes Cancer?
54

 Cancer is caused by
alterations or mutations
in the genetic code
 Can be induced in
somatic cells by:
 Carcinogenic

chemicals
 Radiation

 Some viruses

 Heredity - 5%
 EARLY EVIDENCE THAT
CHEMICALS CAUSE
55
CANCER
• 1775 scrotal cancer &
soot exposure in
English Chimney
Sweepers.
 Catholic nuns - Increased susceptibility to
breast cancer
56
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 Indirect-Reacting
Carcinogens
58

• metabolic conversion
• Procarcinogen
Ultimate carcinogen e.g.
Benzo[a] pyrene from tobacco
smoke and causes lung cancer.
• PAH present in animal fats, in
broiling meat/present in smoked
meats and fish.
• Form epoxides which form
covalent adducts with
macromolecules in the cell
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 MOLECULAR BASIS OF
CANCER
60

 i) Non-lethal genetic damage e.g. environmental

agents such as chemicals, radiation, viruses,
inheritance.

 Clonal expansion of a single progenitor cell that

has incurred the genetic damage.
 Genetically altered cell

61

 Body cells replicate through mitosis, they respond to their

surrounding cells and replicate only to replace other cells.

 A genetic mutation may cause a cell and its descendants

to reproduce even though replacement cells are not
needed.

 A mutation that causes the cell to replicate even though

this tissue does not need replacement cells at this time or
at this place.
 Targets of carcinogens
62

a) Growth -promoting proto-oncogenes-

Genes involved in normal transmission of
growth-promoting signals from the cell surface to
the nucleus (e.g. ras, src) or are transcription
factors (e.g. myc, fos)

b) Growth-inhibiting cancer suppresser

genes.- “tumour suppresser genes”.
both copies of the gene (e.g. Rb, p53,BRCA1 or
BRCA2) must be either lost, mutated or
inactivated.
 Functions of Cellular Proto-Oncogenes

63 1. Secreted Growth Factors

2. Growth Factor Receptors

4. Nuclear
Proteins:
Transcription
3. Cytoplasmic Factors
Signal Transduction
Proteins
5. Cell Growth
Genes
63
 Oncogenes
64

proto-oncogene = ras
Oncogene = mutated ras
Always activated
Always stimulating
proliferation
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 TUMOR SUPPRESSOR GENES
66
Disorders in which gene is affected

Gene (locus) Function Familial Sporadic

DCC (18q) cell surface unknown colorectal

interactions cancer

WT1 (11p) transcription Wilm’s tumor lung cancer

Rb1 (13q) transcription retinoblastoma small-cell lung

carcinoma

p53 (17p) transcription Li-Fraumeni breast, colon,

syndrome & lung cancer

BRCA1(17q) transcriptional breast cancer breast/ovarian

tumors
BRCA2 (13q) regulator/DNA repair
66
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 Cancer Incidence and
68 Death Rate
 Top three cancers
69 Zimbabwe National Cancer Registry (2006)

Men % Women % Boys % Girls %

Kaposi’s 19.9% Cervical 29.4% Retinoblast 18% Wilm’s Tumour 14.5%,
sarcoma cancer oma
Prostate 12.9% Breast 12.9% Non 16.9% Kaposi 12.9%
cancer cancer Hodgkin sarcoma
Non Kaposi’s 10,8% Lymphoma 16.9% Retinoblastoma 12.9%
melanoma of 11.6% sarcoma
the skin
 Factors Believed to Contribute to
70
Global Causes of Cancer
 Risks For Cancer
71

 Lifetime risk – 25% the probability that an

individual, over the course of a lifetime, will
develop cancer or die from it
 Relative risk – measure of the strength of the
relationship between risk factors and a
particular cancer
 Smoking – 30% of all cancer deaths, 87% of
lung cancer deaths
 Obesity – 50% higher risk for breast cancer in
postmenopausal women, 40% higher risk in
colon cancer for men
72

Pap smear, neoplastic cells with large nuclear/cytoplasmic ratios

Invasive squamous cell carcinoma, trachea of a hamster given benze(a)pyrene

 Multistage Carcinogenesis
73

i) Initiation- a permanent change in DNA of target

cell.

ii) Promotion- epigenetic events that selectively

influence the proliferation of the initiated cell.
PROMOTERS
• augment carcinogenicity of some chemicals
NB. many carcinogens have no requirement for
promoting agents.

• iii) Progression- stepwise development of a

cancer cell leading to malignancy and metastatic
potential.
 CARCINOGENESIS
74
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The photo on the right shows a carcinoma of the uterine cervix that is
just beginning to invade into underlying tissue (at the point indicated by
the green arrow). The photo on the left shows the corresponding healthy
tissue, for comparison.
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 Current strategies to combat
cancers
79

• Mechanics -- surgery,1600BC
• Physics -- radiotherapy,1896
• Chemistry -- chemotherapy,1942
• Biology -- immunotherapy,1976
 CELLULAR AND MOLECULAR
80
BASIS OF CHEMOTHERAPY
Alkylating agent:
electron-deficient and will bind to electron-
deficient and will bind to electron-rich groups of
biologic molecules. Mechanism of action:
interaction with bases in DNA + RNA

Antimetabolites: resemble normal metabolites

and compete as substrates for enzyme activity
e.g. methotrexate -an analogue of the vitamin
folic acids
 Inhibits nucleic acid synthesis either directly or
indirectly
 active mainly against proliferating cells.
81