Genotoxicity and carcinogenicity of drugs

Question 1

Terms in Carcinogenesis
carcinogenesis
cancer
carcinogens
tumor
genotoxic

Answer 1

Carcinogenesis: new or autonomous growth of tissue
(neoplasia) resulting in formation of a lesion named
neoplasm
Cancer: malignant neoplasm, disease characterized by
mutation, modified gene expression, cell proliferation, and
aberrant cell growth
Carcinogens: physical or chemical agent that causes or
induces neoplasia
Tumor: lesion characterized by swelling or increase in size,
may or may not be neoplastic
Genotoxic: an agent or process that interacts with cellular
DNA, resulting in alteration of DNA structure

Question 2

Cellular basis of cancer

Answer 2

• Uncontrolled and
  inappropriate division of
  cells
• Genetic damage to critical
  genes that regulate cell
  growth
• Exposure to chemical
  carcinogens considered
  important

Question 3

Where are we exposed to
carcinogens ?

Answer 3

diet (35%)
viruses
smoking
environment
radiation
All of the time…..

Question 4

What factors influence the development of cancer?What factors influence the development of cancer?

Answer 4

Dose, amount and length of exposure. The lower the
dose the least likely you are to develop cancer or related
diseases.
* Environmental or “lifestyle” factors.
* Cigarette smoking (co-carcinogen)
* Alcohol consumption (co-carcinogen)
* Diet, high fat consumption, natural antioxidants
* Geographic location, industrial areas, UV light
* Therapeutic drugs, some are known carcinogens
* Inherited conditions

Question 5

Human carcinogens - Environmental

Answer 5

Aflatoxins Fungus, contaminated peanut and grain
* Asbestos
* Benzene
* Cadmium
* Coal tar
* Creosote
* DDT
* Polycyclic aromatic hydrocarbons
* Radon
* Solar radiation

Question 6

Human carcinogens - Drugs/Therapeutic agents

Answer 6

• Adriamycin (doxorubicin)
• Androgenic steroids
• Chlorambucil
• Cisplatin
• Cyclophosphamide
• Cyclosporin A
• Diethylstilbestrol
• Ethylene oxide
• Melphalan
• Tamoxifen

Question 7

How is chemical carcinogenicity determined?

Answer 7

Epidemiological studies determine the relationship
between a cancer suspect chemical and a human
population over a long period of time.
* Animal studies directly induce cancer in test animals
using a large sample of animals, usually of two or more
species with varying dose and time parameters.
* Experiments with animals are based on the premise that
chemicals that produce cancer in animals will have
similar effects on human cells. Most known human
carcinogens produce cancer in experimental animals.

Question 8

The process of carcinogenesis

Answer 8

Initiation
(irreversible)
malignant
metastases
More
mutations
Progression
(irreversible)
Promotion
(reversible)

Question 9

Initiation Stage

Answer 9

First stage of cancer process
* Rapid
* Irreversible
* First step: DNA modification by initiator (binding of carcinogen or its
functional group)
* Middle step: single DNA replication without previous or simultaneous DNA
repair
* Final step: formation of stable heritable changes in the cell DNA=
mutations

Question 10

Promotion

Answer 10

Epigenetic event, change in gene expression without
change in DNA. Promotion is reversible.
* Mitogenic (Not mutagenic). Stimulates proliferation.
Causes both mutated and normal cells to proliferate.
* Enhances the effect of the genotoxic initiating agent by
establishing clones of initiated cells.
* Long delay possible between administration of initiating
agent and promoting agent.
* Examples: ROS and redox active xenobiotics and
metals, phorbol esters (e.g. TPA), polycyclic aromatic
compounds (e.g. Dioxin), peroxisome proliferators
(oxidized fats), endocrine disruptors (e.g. estradiol,
diethylstilbestrol)

Question 11

Progresssion Stage

Answer 11

• Third (final) stage of the carcinogenesis process
• Conversion of the benign preneoplastic lesions into a neoplastic cancer
• Result of occurrence of genotoxic events inducing chromosomal
  damage such as aberrations and translocations
• Mediated by increase in DNA synthesis and cell proliferation in
  the preneoplastic lesions (promotion stage)
  Chromosome aberrations: breaks and structural changes.
  Examples of progressor agents: inflammation, asbestos fibers,
  benzene, benzoyl peroxide, other peroxides, oxidative stress,
  inflammation

Question 12

Chemical carcinogens

Answer 12

Genotoxic
* Possess the ability to damage DNA
* Can lead to mutations in critical genes resulting in
development of cancer
* Majority of known human carcinogens are genotoxic
Non Genotoxic (epigenetic)
* Don’t damage DNA
*Alter the balance between cellular growth and death
* Lots of non genotoxic chemicals positive in animal
studies

Question 13

Carcinogens classification with regards to their
mode of action

Answer 13

• Primary (direct acting) carcinogens: no need for
  metabolic activation to act as cancer causative agent
• Secondary carcinogens (indirect acting): have
  to be metabolically activated
• Co-carcinogens: increase carcinogenic
  potential by stimulating biotransformation of a
  certain substance
• Promotors: suppress apoptosis; stimulate proliferation

Question 14

Direct Acting Carcinogens

Answer 14

Direct-acting agents require no metabolic conversion to
become carcinogenic. Have highly reactive eletrophile
groups that directly damage DNA, leading to mutations
and eventually cancer.
* Examples are: cancer chemotherapeutic drugs (e.g.
alkylating agents)
* Used as Rx of e.g. leukemia, lymphoma, Hodgkin
lymphoma, and ovarian carcinoma, non-neoplastic
disorders, such as rheumatoid arthritis or Wegener
granulomatosis.
* May evoke later a second form of cancer, usually
leukemia

Question 15

Indirect-Acting Carcinogens

Answer 15

The designation indirect-acting agent refers to chemicals
that require metabolic activation and conversion to an
ultimate carcinogen before they become active.
* Indirect-acting agents are not active until converted to an
ultimate carcinogen by endogenous metabolic pathways
e.g. endogenous enzymes like cytochrome P450
oxygenase.
* Examples: Benzopyrene, polycyclic hydrocarbons,
aromatic amines and Azo dyes, Aflatoxin B1,
insecticides, fungicides, nitrites used as food
preservatives.

Question 16

Induction of metabolizing enzymes

Answer 16

May be a reason for tissue-, and/or species-selectivity of
carcinogens
* Metabolites may be ligands for receptors
* Production of reactive oxygen species

Question 17

Chemical carcinogens

Answer 17

The direct-acting carcinogens interact with
macromolecules through the covalent bond formation
between an electrophilic form of the carcinogen and the
nucleophilic sites in proteins (e.g. S, O, and N atoms in
cysteine, tyrosine, and histidine, respectively) and nucleic
acids (e.g. N and O atoms in purine or pyrimidine), such
as N-methyl-N-nitrosourea, a chemically-reactive alkylating
agent.
* Some agents can intercalate into the DNA double helix by
forming tight noncovalent bonds (e.g. daunorubicin).
* Most of carcinogens are indirectly-acting. Thus, metabolic
activation of certain carcinogenic agents is necessary to
produce the “ultimate carcinogen” that actually reacts with
crucial molecules in target cells

Question 18

Metabolic activation of aflatoxin

Answer 18

Aflatoxin B1, a toxin from a mold (Aspergillus flavus oryzae) that grows on
grain and peanuts when they are stored under humid tropical conditions. It is
thought to be a contributory cause of liver cancer in the tropics

Question 19

Metabolic activation of Tamoxifen

Answer 19

ok

Question 20

Most Frequently Attacked Nucleophilic Centers in DNA

Answer 20

Adduct formation - N7 of adenine and guanine, C8 of guanine; polycyclic
aralkylated molecules on exocyclic N6 of adenine or N2 guanine → substitution
Alkylations – exocyclic O6 of guanine and O4 thymine
N1 adenine or N3 cytosine

Beside molecules that covalently bind to DNA
bases, mutations may be induced, and
carcinogenesis initiated, by agents that interact
with DNAby Van der Waals forces intercalating
between two base-pairs.
Example: doxorubicin, ethydium bromide

Question 21

Beside molecules that covalently bind to DNA
bases, mutations may be induced, and
carcinogenesis initiated, by agents that interact
with DNAby Van der Waals forces intercalating
between two base-pairs.
Example: doxorubicin, ethydium bromide

Answer 21

• An insertion of the flat planar rings of a polycyclic
  hydrocarbon between the stacked bases of doublehelical DNA may distort the helix, leading to a frameshift
  mutation during DNA replication past the point of the
  intercalation.
• Alkylated bases in DNA can mispair with the wrong base
  during DNA replication. For example, O6 methyguanine
  pairs with thymine instead of cytosine during DNA
  replication, leading to a base transition (i.e. GC→AT)
  type of mutation during the next round of DNA
  replication

Question 22

Potential biological consequences of DNAadduct formation

Answer 22

Many of the base adducts formed by carcinogens involve
modifications of N-3 or N-7 positions on purines that
induce an instability in the glycosidic bond between the
purine base and deoxyribose. This destabilized structure
can then undergo cleavage by DNA glycosylase,
resulting in loss of the base.
* Interaction with some carcinogens has been shown to
favor a conformational transition of DNA from its usual
double-helical B form to a Z-DNA form. This could alter
the transcribability of certain genes, since B→Z
conformational transitions are thought to be involved in
regulating chromatin structure.

Question 23

Testing for genotoxicity/carcinogenicity

Answer 23

Tiered approach
* In vitro: e.g. AMES test, mouse lymphoma assay
* Short term in vivo tests: e.g mouse micronucleus assay,
sister chromatid exchange (SCE) assay
* Long term (2 – 3 year) in vivo carcinogenicity assays