BMS67 – Chemical mutagenesis and carcinogenesis
BMS67 – CHEMICAL MUTAGENESIS
AND CARCINOGENESIS
Opleiding: Master Biomedical Sciences
Onderwijsinstelling: Radboud
1
, BMS67 – Chemical mutagenesis and carcinogenesis
Content
Carcinogenesis...................................................................................................................................4
Multi-stage carcinogenesis model........................................................................................................................4
Genotoxic mechanisms.........................................................................................................................................4
Direct-acting carcinogens................................................................................................................................4
Indirect-acting carcinogens.............................................................................................................................5
Mutagenesis....................................................................................................................................................5
DNA repair.......................................................................................................................................................6
Non-genotoxic mechanisms..................................................................................................................................6
Classes and mode of action of non-genotoxic carcinogens............................................................................6
Tumor suppressor gene P53..................................................................................................................................7
Oncogene Ras........................................................................................................................................................7
Wnt signaling.........................................................................................................................................................8
Ros production......................................................................................................................................................8
Chemical carcinogens............................................................................................................................................9
Polyaromatic hydrocarbons (PAHs).................................................................................................................9
Aromatic amines/amides..............................................................................................................................10
Aminoazodyes...............................................................................................................................................10
N-nitroso compounds....................................................................................................................................10
Carbamates...................................................................................................................................................10
Halogenated compounds..............................................................................................................................11
Natural carcinogens......................................................................................................................................11
Metals............................................................................................................................................................11
Anticancer drugs............................................................................................................................................11
Alkylating agents...........................................................................................................................................11
Dioxin (TCDD)................................................................................................................................................11
Biotransformation...........................................................................................................................11
Phase I reactions.................................................................................................................................................12
Phase II reactions................................................................................................................................................12
Induction and suppression of biotransforming enzymes....................................................................................13
Clearance.............................................................................................................................................................13
Short term test principles................................................................................................................13
Ames test.............................................................................................................................................................13
2
, BMS67 – Chemical mutagenesis and carcinogenesis
Mouse lymphoma assay (MLA)...........................................................................................................................13
Micronucleus assay.............................................................................................................................................14
Chromosome aberration test..............................................................................................................................14
Comet assay........................................................................................................................................................14
H(G)PRT assay......................................................................................................................................................14
ToxTracker assay.................................................................................................................................................15
Germ cell mutagens.........................................................................................................................15
Germ cell mutagen assessment..........................................................................................................................15
Big Blue® mouse/rat......................................................................................................................................15
Muta™Mouse................................................................................................................................................15
Risk assessment...............................................................................................................................15
Carcinogenicity studies........................................................................................................................................16
Predictive value of a genotoxicity assay........................................................................................................16
Classification systems for carcinogens................................................................................................................16
Derivation of acceptable human standards........................................................................................................16
Effects with a threshold.................................................................................................................................16
Effects without a threshold...........................................................................................................................17
Human biomonitoring.....................................................................................................................17
Biomarkers and kinetics......................................................................................................................................17
Biomarkers of exposure................................................................................................................................17
Biomarkers of effect......................................................................................................................................17
Choice of bio matrix......................................................................................................................................18
Benzene...........................................................................................................................................18
Toxicokinetics and biotransformation of benzene.............................................................................................18
Mechanisms of action and adverse effects of benzene......................................................................................18
Biomarkers..........................................................................................................................................................19
Risk assessment...................................................................................................................................................19
3
, BMS67 – Chemical mutagenesis and carcinogenesis
Carcinogenesis
Chemical carcinogenesis: a scientific discipline studying the role of chemical factors in the development of
cancer.
Chemical mutagenesis: a scientific discipline studying the role of chemical factors in the development of
hereditary malformations.
↪ A mutagen is always toxic or growth inhibitory at high concentrations.
Multi-stage carcinogenesis model
Initiation: The initiation phase begins when a normal cell
undergoes a permanent genetic alteration, typically due to
exposure to carcinogens such as chemicals, radiation, or
viruses. This alteration usually involves mutations in critical
genes that control cell growth, repair, or apoptosis.
Importantly, initiation itself does not immediately result in
cancer; it merely produces an “initiated cell” that carries the
potential for malignant transformation.
Promotion: In the promotion phase, the initiated cell is
stimulated to proliferate. This stage is not directly caused by
DNA damage but by agents or conditions that favor cell
division and survival, such as chronic inflammation, hormones, or dietary factors. Promotion is reversible in its
early stages, and without continued stimulation, initiated cells may not progress toward malignancy. However,
sustained promotion leads to the expansion of the initiated cell population, thereby increasing the likelihood of
further mutations.
Progression: The progression phase marks the transition to malignancy. During this stage, the proliferating cell
population accumulates additional genetic and epigenetic changes, leading to increasing genetic instability, loss
of normal growth regulation, and acquisition of invasive and metastatic capabilities.
For regulatory purposes, several attempts have been made to characterize chemical carcinogens based on
cellular and molecular pathways that are perturbed following agent exposure:
1. Is electrophilic or can be metabolically activated
2. Is genotoxic
3. Alters DNA repair or causes genomic instability
4. Induces epigenetic alterations
5. Induces oxidative stress
6. Induces chronic inflammation
7. Is immunosuppressive
8. Modulates receptor-mediated effects
9. Causes immortalization
10. Alters cell proliferation, cell death, or nutrient supply
Genotoxic mechanisms
Genotoxic carcinogens are those agents that interact with DNA to
damage or change its structure. They are frequently mutagenic in a
dose-responsive manner, and for regulatory purposes they are
classified as exhibiting low-dose linear patterns, which approximates a
straight line at very low doses without a threshold and a lack of
reversibility of effect upon removal of the agent. DNA-reactive
carcinogens can be further subdivided according to whether they are
active in their parent form and those that require metabolic activation.
Direct-acting carcinogens
A class of carcinogens that do not require metabolic activation or
modification to induce cancer are termed direct-acting or activation-
4
, BMS67 – Chemical mutagenesis and carcinogenesis
independent carcinogens. These chemicals are also defined as ultimate carcinogens. Examination of the
chemical structure of these chemicals reveals that they are highly reactive electrophilic molecules that can
interact with and bind to nucleophiles such as cellular macromolecules including DNA. Generally, these
chemicals containing these moieties frequently cause tumor formation at the site of chemical exposure.
Indirect-acting carcinogens
Indirect-acting carcinogens are compounds that require metabolism in order to react with DNA. It has since
been shown that the majority of DNA-reactive carcinogens are found as parent compounds, or procarcinogens.
Procarcinogens are stable chemicals that require subsequent metabolism to be carcinogenic. It is important to
note that besides activation of the procarcinogen to a DNA-reactive form, detoxification pathways may also
occur. Indirect-acting genotoxic carcinogens usually produce their neoplastic effects, not at the site of exposure
but at the target tissue where the metabolic activation of the chemical occurs.
Mutagenesis
The reaction of a carcinogen with genomic DNA either directly or indirectly may result in DNA adduct formation
or DNA damage and frequently produces a mutation. These events are dependent upon when in the cell cycle
the adducts are formed, where the adducts are formed, and the type of repair process used in response to the
damage. Transitions are a substitution of one pyrimidine (C, T, U) by the other or one purine (A, G) by the other
(changes within a chemical class), whereas a transversion occurs when a purine is replaced by a pyrimidine or a
pyrimidine is replaced by a purine (changes across a chemical class). Carcinogens can induce transitions and
transversions in several ways:
When adducts (or apurinic or apyrimidinic sites) are encountered by the DNA replication processes,
they may be misread. The polymerase may preferentially insert an adenine (A) in response to a
noninformative site. Thus, the daughter strand of an A, C, G, or T adduct will have an adenine (A) and
this change is fixed (mutation) and resistant to subsequent DNA repair.
A shift in the reading frame (resulting in a frame shift mutation). Most frame shift mutations are
deletions and occur more frequently when the carcinogen–DNA adduct is formed on a nucleotide.
DNA strand breaks can also result from carcinogen–DNA adducts. These may arise either because of
excision repair mechanisms that are incomplete during DNA replication or via direct alkylation of the
phosphodiester backbone leading to backbone cleavage. Strand scission can lead to double-strand
breaks, recombination, or loss of heterozygosity.
To study at the DNA level whether exposure to chemicals will lead to tumor formation in a certain tissue, cells
or test animals are exposed to carcinogenic substances. This shows that the places in the DNA where adducts
can be detected are also the places that are frequently mutated. If the same mutation pattern (location and
type of mutation) is subsequently found in human tumor tissue, this is a clear indication that the tumor was
created by exposure to the carcinogen in question.
DNA adduct analysis can be carried out with several techniques:
32P-Postlabeling DNA is enzymatically digested into 3’-
monophosphate nucleotides using nucleases. The nucleotides
containing adducts are tagged at their 5’-end with radioactive
phosphate (32P) using the enzyme T4 polynucleotide kinase. The
labeled adducts are separated by thin-layer chromatography
(TLC) or high-performance liquid chromatography (HPLC).
Immunoassay (e.g. ELISA, IHC) This relies on antibodies that
recognize specific DNA modifications.
Gas chromatography-mass spectrometry (GC/MS) and liquid
chromatography-mass spectrometry (LC/MS) DNA is first
isolated and hydrolyzed to release nucleosides or nucleotides.
Because DNA adducts are typically non-volatile, they require
chemical derivatization, such as silylation, to make them suitable for gas chromatography. The
derivatized adducts are then separated in the GC column and detected by the mass spectrometer,
which provides detailed structural information about the adducts.
5
BMS67 – CHEMICAL MUTAGENESIS
AND CARCINOGENESIS
Opleiding: Master Biomedical Sciences
Onderwijsinstelling: Radboud
1
, BMS67 – Chemical mutagenesis and carcinogenesis
Content
Carcinogenesis...................................................................................................................................4
Multi-stage carcinogenesis model........................................................................................................................4
Genotoxic mechanisms.........................................................................................................................................4
Direct-acting carcinogens................................................................................................................................4
Indirect-acting carcinogens.............................................................................................................................5
Mutagenesis....................................................................................................................................................5
DNA repair.......................................................................................................................................................6
Non-genotoxic mechanisms..................................................................................................................................6
Classes and mode of action of non-genotoxic carcinogens............................................................................6
Tumor suppressor gene P53..................................................................................................................................7
Oncogene Ras........................................................................................................................................................7
Wnt signaling.........................................................................................................................................................8
Ros production......................................................................................................................................................8
Chemical carcinogens............................................................................................................................................9
Polyaromatic hydrocarbons (PAHs).................................................................................................................9
Aromatic amines/amides..............................................................................................................................10
Aminoazodyes...............................................................................................................................................10
N-nitroso compounds....................................................................................................................................10
Carbamates...................................................................................................................................................10
Halogenated compounds..............................................................................................................................11
Natural carcinogens......................................................................................................................................11
Metals............................................................................................................................................................11
Anticancer drugs............................................................................................................................................11
Alkylating agents...........................................................................................................................................11
Dioxin (TCDD)................................................................................................................................................11
Biotransformation...........................................................................................................................11
Phase I reactions.................................................................................................................................................12
Phase II reactions................................................................................................................................................12
Induction and suppression of biotransforming enzymes....................................................................................13
Clearance.............................................................................................................................................................13
Short term test principles................................................................................................................13
Ames test.............................................................................................................................................................13
2
, BMS67 – Chemical mutagenesis and carcinogenesis
Mouse lymphoma assay (MLA)...........................................................................................................................13
Micronucleus assay.............................................................................................................................................14
Chromosome aberration test..............................................................................................................................14
Comet assay........................................................................................................................................................14
H(G)PRT assay......................................................................................................................................................14
ToxTracker assay.................................................................................................................................................15
Germ cell mutagens.........................................................................................................................15
Germ cell mutagen assessment..........................................................................................................................15
Big Blue® mouse/rat......................................................................................................................................15
Muta™Mouse................................................................................................................................................15
Risk assessment...............................................................................................................................15
Carcinogenicity studies........................................................................................................................................16
Predictive value of a genotoxicity assay........................................................................................................16
Classification systems for carcinogens................................................................................................................16
Derivation of acceptable human standards........................................................................................................16
Effects with a threshold.................................................................................................................................16
Effects without a threshold...........................................................................................................................17
Human biomonitoring.....................................................................................................................17
Biomarkers and kinetics......................................................................................................................................17
Biomarkers of exposure................................................................................................................................17
Biomarkers of effect......................................................................................................................................17
Choice of bio matrix......................................................................................................................................18
Benzene...........................................................................................................................................18
Toxicokinetics and biotransformation of benzene.............................................................................................18
Mechanisms of action and adverse effects of benzene......................................................................................18
Biomarkers..........................................................................................................................................................19
Risk assessment...................................................................................................................................................19
3
, BMS67 – Chemical mutagenesis and carcinogenesis
Carcinogenesis
Chemical carcinogenesis: a scientific discipline studying the role of chemical factors in the development of
cancer.
Chemical mutagenesis: a scientific discipline studying the role of chemical factors in the development of
hereditary malformations.
↪ A mutagen is always toxic or growth inhibitory at high concentrations.
Multi-stage carcinogenesis model
Initiation: The initiation phase begins when a normal cell
undergoes a permanent genetic alteration, typically due to
exposure to carcinogens such as chemicals, radiation, or
viruses. This alteration usually involves mutations in critical
genes that control cell growth, repair, or apoptosis.
Importantly, initiation itself does not immediately result in
cancer; it merely produces an “initiated cell” that carries the
potential for malignant transformation.
Promotion: In the promotion phase, the initiated cell is
stimulated to proliferate. This stage is not directly caused by
DNA damage but by agents or conditions that favor cell
division and survival, such as chronic inflammation, hormones, or dietary factors. Promotion is reversible in its
early stages, and without continued stimulation, initiated cells may not progress toward malignancy. However,
sustained promotion leads to the expansion of the initiated cell population, thereby increasing the likelihood of
further mutations.
Progression: The progression phase marks the transition to malignancy. During this stage, the proliferating cell
population accumulates additional genetic and epigenetic changes, leading to increasing genetic instability, loss
of normal growth regulation, and acquisition of invasive and metastatic capabilities.
For regulatory purposes, several attempts have been made to characterize chemical carcinogens based on
cellular and molecular pathways that are perturbed following agent exposure:
1. Is electrophilic or can be metabolically activated
2. Is genotoxic
3. Alters DNA repair or causes genomic instability
4. Induces epigenetic alterations
5. Induces oxidative stress
6. Induces chronic inflammation
7. Is immunosuppressive
8. Modulates receptor-mediated effects
9. Causes immortalization
10. Alters cell proliferation, cell death, or nutrient supply
Genotoxic mechanisms
Genotoxic carcinogens are those agents that interact with DNA to
damage or change its structure. They are frequently mutagenic in a
dose-responsive manner, and for regulatory purposes they are
classified as exhibiting low-dose linear patterns, which approximates a
straight line at very low doses without a threshold and a lack of
reversibility of effect upon removal of the agent. DNA-reactive
carcinogens can be further subdivided according to whether they are
active in their parent form and those that require metabolic activation.
Direct-acting carcinogens
A class of carcinogens that do not require metabolic activation or
modification to induce cancer are termed direct-acting or activation-
4
, BMS67 – Chemical mutagenesis and carcinogenesis
independent carcinogens. These chemicals are also defined as ultimate carcinogens. Examination of the
chemical structure of these chemicals reveals that they are highly reactive electrophilic molecules that can
interact with and bind to nucleophiles such as cellular macromolecules including DNA. Generally, these
chemicals containing these moieties frequently cause tumor formation at the site of chemical exposure.
Indirect-acting carcinogens
Indirect-acting carcinogens are compounds that require metabolism in order to react with DNA. It has since
been shown that the majority of DNA-reactive carcinogens are found as parent compounds, or procarcinogens.
Procarcinogens are stable chemicals that require subsequent metabolism to be carcinogenic. It is important to
note that besides activation of the procarcinogen to a DNA-reactive form, detoxification pathways may also
occur. Indirect-acting genotoxic carcinogens usually produce their neoplastic effects, not at the site of exposure
but at the target tissue where the metabolic activation of the chemical occurs.
Mutagenesis
The reaction of a carcinogen with genomic DNA either directly or indirectly may result in DNA adduct formation
or DNA damage and frequently produces a mutation. These events are dependent upon when in the cell cycle
the adducts are formed, where the adducts are formed, and the type of repair process used in response to the
damage. Transitions are a substitution of one pyrimidine (C, T, U) by the other or one purine (A, G) by the other
(changes within a chemical class), whereas a transversion occurs when a purine is replaced by a pyrimidine or a
pyrimidine is replaced by a purine (changes across a chemical class). Carcinogens can induce transitions and
transversions in several ways:
When adducts (or apurinic or apyrimidinic sites) are encountered by the DNA replication processes,
they may be misread. The polymerase may preferentially insert an adenine (A) in response to a
noninformative site. Thus, the daughter strand of an A, C, G, or T adduct will have an adenine (A) and
this change is fixed (mutation) and resistant to subsequent DNA repair.
A shift in the reading frame (resulting in a frame shift mutation). Most frame shift mutations are
deletions and occur more frequently when the carcinogen–DNA adduct is formed on a nucleotide.
DNA strand breaks can also result from carcinogen–DNA adducts. These may arise either because of
excision repair mechanisms that are incomplete during DNA replication or via direct alkylation of the
phosphodiester backbone leading to backbone cleavage. Strand scission can lead to double-strand
breaks, recombination, or loss of heterozygosity.
To study at the DNA level whether exposure to chemicals will lead to tumor formation in a certain tissue, cells
or test animals are exposed to carcinogenic substances. This shows that the places in the DNA where adducts
can be detected are also the places that are frequently mutated. If the same mutation pattern (location and
type of mutation) is subsequently found in human tumor tissue, this is a clear indication that the tumor was
created by exposure to the carcinogen in question.
DNA adduct analysis can be carried out with several techniques:
32P-Postlabeling DNA is enzymatically digested into 3’-
monophosphate nucleotides using nucleases. The nucleotides
containing adducts are tagged at their 5’-end with radioactive
phosphate (32P) using the enzyme T4 polynucleotide kinase. The
labeled adducts are separated by thin-layer chromatography
(TLC) or high-performance liquid chromatography (HPLC).
Immunoassay (e.g. ELISA, IHC) This relies on antibodies that
recognize specific DNA modifications.
Gas chromatography-mass spectrometry (GC/MS) and liquid
chromatography-mass spectrometry (LC/MS) DNA is first
isolated and hydrolyzed to release nucleosides or nucleotides.
Because DNA adducts are typically non-volatile, they require
chemical derivatization, such as silylation, to make them suitable for gas chromatography. The
derivatized adducts are then separated in the GC column and detected by the mass spectrometer,
which provides detailed structural information about the adducts.
5
