Teratogenicity and Carcinogens

By: Sagni Hanbisa

B-pharm, MSc in
Pharmacology
Wallaga University

1
 Mutations
What is mutation ?
 Mutations are unexpected and undirected changes
in the composition of genetic information
 It may be qualitative and quantitative changes in
the information structure of the individual gene
and the genome
 It forms the basis of the development of neoplasia
2
Arise

– Spontaneuosly
– External factors
• Temprature
• Chemical substances
• UV and Ionizing radiation
 Mutation can occur in somatic cells as well as in
reproductive cells
 Mutations taking place on the reproductive cells, are
transmited to the offispring 3
 Mutations happen regularly

 Many mutations are repaired by enzymes

4
 Types of mutation

1. Single-point mutations or gene mutations

2. Structural chromosomal aberrations
3. Genomic mutations or aneuploidy

5
 1. Gene mutation
Base-pair substitutions
Transition
Transversion

 Molecular mechanisms causing

– Incorporation of base analogue

– Chemical alteration of normal base

– Binding of chemical to bases

– Spontaneous base modification

6
Incorporation of base analogue

– During replication of DNA, base analogs

can be incorporated at the site of the
correct bases
– A-T to G-C transition

• 5-bromouracil (5-BU), in shortage of

thiamine 7
8
Chemical changes in normal bases

– Bases can be altered

• Hydroxylamine

• Nitrous acid

– Treatment of Adenine (A) with HNO2 causes deamination of A,

hypoxanthine is formed, which behaves like guanine.

• A-T to G-C transition

– Hydroxylamine only react with pyrimidines in the DNA

• C-G to T-A
9
10
Binding of chemicals to bases

– Base-pair substitutions can also be achieved by alkylation of

the bases

– Alkylating chemical substances constitute the largest group of

mutagenic agent

– Highly potent alkylating agents are

• Mustard gas
• Dimethyl sulfonic acid
• Diethyl sulfonic acid
• Methyl methane sulfonic acid
• epoxides 11
 Mutation may also be induced by binding of
relatively large compound to the DNA bases
resulting in so called ‘’bulky adducts’’
– Benz [a] pyrene (BP) metabolite 7,8-dihydrodiol-
9,10-epoxide is major mutagenic and carcinogenic
product
• GC – TA transversion
12
Spontaneous base modification
Although the DNA bases are chemically
stable, it is possible that under the
physiological condition certain
spontaneous modifications occur
• Spontaneous deamination of cytosine

13
14
 Addition and deletion of bases

• May arise spontaneously or may be caused by mutagenesis

– Also known as frameshift mutation

• When substitution affect a single codon, deletion or

addition changes all codons following the site of mutation

• Chemicals substances which cause frameshift mutation are

usually characterized by large molecules
Eg Acridines
proflavin
15
 2. Structural chromosomal aberrations

 Chromosomal aberration means that a piece of

chromosomal material (i.e. genetic information)
ends up at a d/t location with in the same or in a d/t
chromosome, is lost or conversely, is present in
excess (amplification)
 Many chromosomal aberrations are lethal to the cell

17
 3. Genome mutation

Involve a change in the number of

chromosomes
Euploidy…multiple of the complete set of
chromosomes
Aneuploidy…numerical change in some
chromosomes and the total number of
chromosomes is not change
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 Aneuploidy is caused by a fault in the cell-division
mechanism.

 Well-known inhibitors of neuclear spindles are

colchicine, vinblastine, vincristine and taxol
(mutagenic)

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 SOURCES OF MUTATIONS
EXOGENOUS DNA
ENDOGENOUS DNA
DAMAGE
DAMAGE
Polymeras Environmenta Lif
Free e l
Depurinatio Agent e
Style
Radicals Errors n s s

DNA REPAIR

CELL REPLICATION

MUTATION
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 Mutagenicity tests
 More than 100 tests are currently available, using a
wide range of organisms
 In vitro tests are carried out employing
bacteria ,bacteriophages, mamalian cells and human
cells
 Fruit flies, mammals and human beings themselves
are being used in invivo tests
21
 Ames test
 A test developed by Bruce Ames to determine if a
chemical is a mutagen
 Bacterial strains of salmonella typhimumrium are used
 These strains have been selected by Ames, and due to a
specific mutation, require histidine in their culture media
for normal growth
 Upon Addition of a mutagenic subs to the medium, the
DNA is mutated at the same locus, these bacteria regain
the capability to produce histidine
– Therefore called back-mutations or reversions 22
The revertants will form visible colonies on
histidine-deficient agar plates with in a few
days

The number of colonies per plate is measure of

or the mutagenic potential of the test compound

It is gene mutation test

23
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 Why the Ames test is so useful ??

 It is fast: requires just a few days to complete

 Can be performed easily
 Many compounds can be tested at one time
 It is very sensitive: can detect one mutation per
billion cells
 Can detect mutagens formed as a result of
normal cellular function

25
 Carcinogenesis
 Chemical carcinogenesis :
– is the study of the process through w/c chemical
carcinogens cause cancer

 Carcinogenesis
-process through w/c cancer develops

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 Key words
– Oncology
– A neoplasm or neoplasia
– Benign neoplasms
– Carcinogen

27
 Cancer
 A subset of neoplastic lesions , forming neoplasm which
is relatively autonomous growth in tissue with abnormal
regulation of gene expression

 malignant type Neoplasm with metastasis: growth type

where secondary growths are developed form the primary
neoplasm

 All have one common thing  caused by uncontrolled

cell division
 Most are monoclonal in origin
 Critical genes

• Proto-oncogenes
 +ve regulators of cell growth
 Frequently mutated in cancer
• Tumor suppressor genes
 Negative regulators of cell replication
 Mutation causes loss of function—make them
inactive
 Both two critical genes mutation also impairs apoptosis-
leads to cancer

29
 The six properties of cancerous cells

1. Disregard external and internal signals that regulate

cell proliferation
2. Tend to avoid suicide by apoptosis
3. Circumvent programmed limitations to
proliferation, escape replicative senescence and
avoid differentiation
4. Genetically unstable
5. Escape from their home tissues (are invasive)
6. survive and proliferate in foreign sites (metastasize)
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31
 Agents Causing Neoplasm
 Carcinogens – substances known to cause cancer or produces

an increase in incidence of cancer in animals or humans

– Cause of most cancers is unknown

– Most cancers are probably multifactorial in origin

• Known carcinogenic agents constitute a small percentage of cases

• Unidentified ‘environmental’ agents probably play a role in 95% of

cancers
32
 Types :-
1. Chemical Oncogensis
2. Radiation Oncogenesis
3. Viral Oncogenesis
4. Nutritional Oncogenesis
5. Hormonal Oncogenesis
6. Genetic Oncogenesis,etc

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 1 - Chemical Carcinogenesis
 Types
1.Proximate or direct-acting : act locally without
metabolic change.
e.g Alkylating Agents
Cyclophosphamide
2.Indirect acting : carcinogenic only after being
metabolised into active compounds (procarcinogen 
ultimate carcinogen)

e. g Polycyclinc hydrocarbons – Benzpyrene

Aromatic amines, dyes - Benzidine
Natural products: Aflotoxin
Others: Vinyl chloride, turpentine etc
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 Pro-carcinogen

Inactivation Activation

Proximate
carcinogen
Detoxication
products

Activation

Ultimate
carcinogen=initiation

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 Steps in Chemical Carcinogenesis
1. Biotransformation
2. Initiation: Covalent binding to DNA
3. Fixation: Mutation stabilized by mitosis
4. Gene expression, transformation
5. Neoplastic growth, proliferation
6. Progression, local effects
7. Metastasis

36
 Mode of carcinogenesis

o Inducing changes in DNA – eg. Base alkylation, deletion,

breakage, cross-linkage
o Synergistic action with viruses

o Promoter for other carcinogens

 Difficulties in identifying carcinogen

o Numerous industrial, agricultural, household chemicals
present in low levels
o Exposed to large number of chemicals in a lifetime

o Long lag phase 37

 2. Radiation Oncogenesis
 Types of oncogenic radiation
– Ultraviolet
– X-ray
– Radioisotopes
– Nuclear Fallout

 Mode of oncogenesis
– Direct effect on DNA
– Activation of cellular oncogenes
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 2. Radiation Oncogenesis cont…
 Ionizing radiation  dysjunction  random
fusion  mutation.
Neoplasia

Mutations

 X Ray workers – Leukemia

 Radio-isotopes – Thyroid carcinoma
 Atomic explosion – Skin cancer, Leukemia
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 2. Radiation Oncogenesis…

 UV Radiation
– Solar UV radiation associated with skin cancers –
squamous CA, basal cell CA, malignant melanoma
– Fair-skinned and elderly are susceptible

– UV light is believed to induce cross-linkages between

DNA molecules and CA occurs when repair
mechanisms are not efficient
40
 2. Radiation Oncogenesis…

 X-ray radiation
– Earlier use of X-rays caused skin cancer, leukemia and
papillary thyroid CA
– Radiotherapy causes raditation-induced malignancy 10-30
yrs later – usually sarcomas
– Diagnostic X-rays are considered to have no increased
risk except in abdominal x-rays which increase incidence
of leukemia in the fetus
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 2. Radiation Oncogenesis…
 Radioisotopes
– Osteosarcoma -common among factory workers who use
radium-containing paints
– associated with lung cancer
– Thorium increases risk of liver cancer – hepatocellular,
angiosarcoma, cholangiocarcinoma
– Radioactive iodine – increased risk of cancer 15-25 years later

 Nuclear Fallout
– Hiroshima, Nagasaki (atomic blasts)
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 3. Viral Oncogenesis
Types
– Oncogenic RNA Viruses
– Oncogenic DNA Viruses
 Insertion of viral nucleic acids  mutation
 Alterations in Oncogenes, cancer suppressor
genes and genes regulating DNA repair:
 resulting in up-regulation of cell division 
Carcinogenesis.

43
 Human Papilloma Virus
– Cervical neoplasia – warts, papilloma cx ca

 Epstein-Barr virus –
– Burkitts Lymphoma, Nasopharyngeal ca.

 Hepatitis B & C virus

– Hepatocellular carcinoma.

 Bacteria, fungi as well as endoparasitic worms can have

role in development of cancer
44
 4. Nutritional Oncogenesis

Scant evidence linking cancer to diet

except for known chemical
carcinogens
Some associations
• Low-fiber diet and colonic CA
• Fatty diet with breast ca

45
 5. Hormonal Oncogenesis
Types
– Induction of Neoplasms by Hormones
– Dependence of Neoplasms on Hormones
Hormones inducing Neoplasms
– Estrogen – breast ca
– Diethylstilbestrol (DES) – vaginal and
uterine ca

46
 Hormonal Dependence of Neoplasms

– Neoplasm not caused by hormones but depend on hormones

for optimal growth
– Neoplastic cells possess receptors for binding hormone
– Loss of hormonal stimulation slow but does not halt growth
– Examples
• Prostate CA
• Breast CA
• Thyroid CA

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48
 6. Genetic Oncogenesis
 Some forms of cancer are related to heredity
(Inheritance)
-Genetic polymorphisms
Examples
– Retinoblastoma
– Wilm’s tumor
– Others
» Neurofibromatosis (type 1 von Recklinghausen’s
disease)
» Multiple endocrine adenomatosis (MEN)
» Familial polyposis coli
» Nevoid basal cell carcinoma syndrome
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 Stages of carcinogenesis

 The pathogenesis of neoplasm consists of three

defined stages
– Initiation
– Promotion
– Progression
 Only in the first and last stages of neoplastic
development , that structural changes in the
genome can be observed , only altered expression
of genes is a characteristic for the stage of
promotion 50
51
 Initiation
• Mutation in relatively specific genes , as
protoncogenes , tumor suppressor genes may be the
most critical step in neoplastic transformation
• It is a Irreversible process in that the genotype / or
phenotype of the cell established at the time of
initiation
• It requires one or more rounds of cell division for the
fixation of the process
• Not all the initiated cells survive over the life span of
the organism due to normal processes of programmed
cell death or DNA repair 52
53
 Promotion
 Promoting agents and their metabolites
generally don’t interact directly with the DNA

 Unlike initiation , promoting agents don’t

involve mutational changes in the genome ,
but reversible alteration of expression of gene
information or acts on cellular components
involved in cell division

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55
 Progression
 Malignant progression is characterized
– At the structural level:
by high rate of growth , invasiveness, Metastasis
– At molecular level:
• Complex genetic alteration as chromosomal
translocation ,deletion , gene amplification resulting to
karyotpic instability that lead to multiple changes in the
malignant cells
– Irreversible changes in the gene expression

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 Risk factors
57

 Age  Life style factors

 Individual genetic make -smoking
up -diet
– Genetic polymorphism -cultural
 Environment -Sexual behavior
– Man made chemicals -occupation
– Life style factors like -radiation
-exposure to substances in
air,H20,etc
 Determination of Carcinogens
 Animal studies
– The lab. Animals are exposed to Carcinogens in
very large doses.

 Epidemiologic studies
– By looking at the factors that might affect the
occurrence of cancer in different populations.

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 Reproductive Toxicology
 Developmental toxicity
 Reproductive toxicology
 Teratology
 Reproductive toxicity
 Teratogenicity
 Teratogen

Define the above terms ???????????

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 Reproductive toxicology :- is the subject area dealing
with cause , mechanisms ,effects and prevention of
disturbances through out the entire reproductive
cycle induced by chemicals

 Developmental toxicology
– The occurrence of adverse effects on the developing
organism that may result from:
• exposure prior to conception (either parent)
• during prenatal development
• postnatally to the time of sexual maturation
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 Mode of action
 Mechnisms
– Mutation
• Chromosomal aberration
– Disturbances in cell division
– Change in nucleic acid composition and protein
synthesis
– Reduction in the amount of essentail constituents
for biosynthesis
– Reduction of the energy supply for embryonic and
fetal d’vt
61
 Mode contd…
– Disturbance of enzyme systems
– Disturbance in regulation of water and electrolyte
balance
– Change in membrane characteristics

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 Pathogenesis
– Increased or decreased cell death
– Disturbance of cell to cell contact
– Reduced biosynthesis
– Increased morphogenetic pattern formation
 Dysmorphogenesis
– Mechanism…
• pathogenesis…dysmorphogenesis…developmental
defect (=effect)

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 Effects
 Functional and postural disorder
• Alcohol and infections by the rubella virus or toxoplasma
 Fertility disorders
 Structural defects or malformations
 Growth retardations
• Alcohol,warfarin,narcotics
 Intrautrine death
• Infections,chemicals,dietry factors and chromosomal
aberration

64
Other effects
– Pharmacological effects during last trimester
• Withdrawal symptoms in neonate caused by
mother’s use of narcotic
• Psychopharmacological drugs or sleeping pills
and, hypotension and hypoglycemia as result of
maternal use of antihypertensive drugs
• Hyperbilirubinemia (jaundice) as a result of
sulphonamide adminstration to the mother

65
 Teratology :- study of abnormal development
(prenatal)
Teratogens :- anything capable of disrupting
embryonic or fetal development and producing
malformations
o birth defects
• usually act during a critical period

Three main principles of teratogenesis

- Dosage
- Time of exposure
- Genotype of the embryo

66
 Fetal development has different stages
1. Blastogenesis (15-21 days )
– Differentiation and germ layer formation period
– Drug therapy has no effect
2. Organogenesis (14-56 days after fertilization )
– Development of major organs
– Drug exposure may lead to congenital
abnormalities(deformity)
3. Fetal period (9th week on wards)
– Drug exposure may cause fetal toxicity rather than
deformity
67
 Principles of teratology
In 1959,James wilson proposed six-
principles of teratology
1. Susceptibility to teratogenesis
depends on the genotype of the
conceptus and the manner in which
this interacts with adverse
environmental factors
68
2. Susceptibility to teratogenesis varies with the
developmental stage at the time of exposure
to an adverse influence
– Preimplantation…resistance or susceptible to
embrytoxicity (embryonic lethality)
– Implantation-----time of organogenesis…highly
susceptibility to dysmorphogenesis
(morphological defect)
– Fetal------neonatal stage…minimal susceptibility
to defect induction) 69
3.Teratological agents act in specific way
(mechanisms) on developing cells and tissues to
initiate sequences of abnormal development
events (pathogenesis)

4. The access of adverse influences to developing

tissues depends on the nature of the influence
(agent)
– Cyclophosphamide (cytotoxic)…emryonic death or
diffuse anatomical abnormalities 70
5. The four manifestations of deviant
development are death , malformation, growth
retardation, and functional defect
• Organogenesis…lethality, growth/functional retardation
and malformation
• Fetal stage…functional deficit and growth retardation

6. Manifestations of deviant development increase

in frequency and degree as dosage increases
from no effect to the totally lethal level
71
Examples of mutagenes,
carcinogenes and teratogenes

72
 Teratogens and their sequalae
 Historical examples

73
 1. Thalidomide
 It had been introduced in 1959 as
– A sedative / hypnotic and
– Ameliorate Nausea/Vomiting in pregnancy
 Following the association with birth defects, it
was withdrawn from the market (1961)

 The affected individuals had

– Amelia (absence of the limbs)
– Phocomelia (reduction of the long bones of the
limbs) 74
75
 Congenital heart disease; occular,intestinal and
renalanomalies ; and malformations of the
external and inner ears were also involved

76
 Thalidomide has recently been approved by
FDA for
– Oral ulcers associated with AIDS
– Erythema nodosum leprosum
– Inflammatory complication of Hansen's disease
(leprosy)

77
 2. Diethylstilbestrol
 DES is synthetic non steroidal estrogen
 Used to prevent threatened miscarriage by
stimulating synthesis of estrogen and
progesterone in the placenta
 In females exposure induced vaginal
adenocarcinoma (transplantal carcinogenesis)
and adenosis and cervical erosion
 In male, hypotrophic testes, hypospadial and
poor semen volume and quality resulted
78
 3. Ethanol

 Cause fetal alcohol syndrome (FAS)

 FAS comprises
• Craniofacial dysmorphism
• Intrauterine and post natal growth retardation
• Retarded psychomotor and intellectual dev’t
• Other non specific major and minor abnormalities
 Mechanism is not well understood but,
complex combination of maternal factors and
biochemical/cellular effects in the embryo
79
80
81
 4. Tobacco smoke

 Consequences include
– Spontaneous abortion
– Prenatal deaths
– Increased risk of sudden infant death syndrome
– Increased risk of learning, behavioral & attention
disorder and lower birth weight
 Nicotine, is a known neuroteratogenic in
experimental animals and can by itself produce
many of the adverse developmental out comes
82
 5. Cocaine

 plenty of adverse effects appear to be

reliably associated with cocaine exposure in
human including
– Premature labor and delivery
– Microencephaly
– Altered pros-encephalic dev’t—decreased
birth weight
– A neuronal neurologic syndrome of abnormal
sleep etc 83
 6. Retinoic acid

 AKA isotretinoin or accutane

 These are vitamine A derivatives and are used
to treat acne
 Therapeutic doses are teratogenic
Causes renal anomalies including hydronephrosis

84
Retinoic Acid

85
 7. Valproic acid (2-propyl pentanoic acid)

 Anti-epileptic drug
 Alter the synthesis of GABA
 Prevent complete closure of the neural tube
 Restrict proliferation in the mid-G1 phase of
the cell cycle and
 Alters the glycosylation state of cell surface
glycoprotiens, w/c cell-matrix interactions
critical to development
86
 8. Cyclophosphamide (CP)
 Used as treatment of lymphocytic leukemia and in
prevention of transplanted organ rejection
 Children born to mothers receiving the drug during 1st
trimester exhibit
– Ectrodactyl (absence of all or parts of digits)
– Cardiac defects
– Decreased fertility
 Must be metabolically activated to be teratogenic
– Acrolein (toxic)
– Phosphoramid mustard (highly toxic)
87
 Testing for teratogenicity
They use at least two common
mammalian laboratory species that are
given several different doses of the test
agent once or several successive days
during organogenesis and early fetal
period
– Coventionally 3 doses are administered; the
highest causing maternal toxicity

• Evaluation of human case reports and

epidemiological investigation (retrospective
and prospective). 88
 Modern safety assessment
 Regulatory guidelines for in vivo testing
 Multigeneration tests
 Epidemiology
 Concordance of data (among species)
 Elements of risk assessment use-in pregnancy rating:
A, B, C, D, X

89
 Tip
 FDA classify drug according to their potential risk
during pregnancy
Category X
 Cause fetal abnormalities in the human and animal
 C/I to women who are or may become pregnant
Eg. Aminopterin
• May induce abortion
• Cause multiple gross anomalies (cleft
lip/palate,short forearms)

90
 Iodinated glycerol
– Cause congenital goiter, hypothyroidism
 Vitamin A and it derivatives
– Heart ,CNS,and facial defects
 Hormones (estrogen,diethylstilbestrol,other
contraceptives)
– Adrenocarcinoma (in female fetus after puberty)
 Vaccines (meales,mumps,rubella vaccines)
– May cause infection of the fetus 91
 Category D
– May have fetal risk but their use in pregnancy is
acceptable in case of life threatening and serous
diseases
 Eg of drugs
– ACE inhibitors
• Renal damage ,hypotension
– Ethanol
• Fetal alcohol withdrawal syndrome and mental
retardation
– Benzodiazepine
• Chronic use may lead to neonatal dependence
92
- Warfarin
• Hypoplastic nasal bridge
• Chondrodysplasia
• CNS malformations
• Risk of bleeding

93
 Category C
Studies in animal have revealed Adverse Drug
Effects on the fetus (teratogenic or embryocidal
or other ) but no controlled studies in human
Drug should be given only if the potential benefit
justifies the potential risk to the fetus
Eg of drugs
• Metronidazole
• Quinolones
• All illicit drugs (heroin, marjuana, LSD,amphetamine)
• others: CAF, ephedrine, prozocin, digoxin
94
 Category B
 There are no controlled studies in pregnant
women or animal-reproduction
Category A
 Controlled studies in women fail to
demonstrate a risk to the fetus in the first
trimester
 There is no evidence of a risk in late trimesters
– The possibility of fetal harm is less likely

95
Mutation, carcinogenesis and teratogenecity
relationship

Although it is known that genotoxic

substances can interfere with cell
defferentiation and there by produce
teratogenic effects

“Most teratogenic substances are not

mutagenic and vice versa’’
96
Chemical which have a mutagenic as
well as teratogenic effect include;
– Cyclophosphamide
– Ethylmethane sulfonic acid
– Methyl methane sulfonic acid
– Busulfan
– NH2
– Triethylene melamine
97
 Chemical Show a high correlation b/n mutagenicity
and carcinogenecity

– Aromatic amines,
– Polycyclic aromatic hydrocarbons
– Nitrosamines
– Alkylating chemicals

 Chemical Show a low correlation b/n mutagenicity

and carcinogenecity

– Hormones
98
–
 Mutagenic non-carcinogens

– NaN2

– Dichlorvos
– Organophosphate

 Non mutagenic carcinogens

– Hormones
– Chemical w/c disturb the hormonal balance

99
 Contrary to mutagenecity and carcinogenecity,
w/c are affected by genetic mechanisms (point
mutation and chromosomal aberration),
– teratogenic effects are usually brought about by non
genetic mechanisms
• Enzyme inhibition

• Altered osmolarity
“Not much is known about the r/n ship b/n
carcinogenicity and teratogenecity”
100
 Recommendations
• Disease have to be treated in all cases.
• We should use drugs with well-known effect on
pregnancy without signs of embryotoxicity.
• It is not recommended to change quickly a lot of
drugs.
• It is not recommended to use combinations of
various drugs. Undesirable effects may be
multiplied.
‘’Any woman in reproductive age may be
pregnant !! So ….’’
101
Thanks!!