Carcinogens

Question 1

Carcinogens

Answer 1

• Carcinogens: chemical or physical sources that initiate or promote carcinogenesis
• Combinations of different carcinogens can increase the risk of tumor formation
• Several risk factors (e.g. working place, smoking, medication, sun burns) are associated with cancer
• Carcinogenic: an exposure that can lead to cancer in humans or animals
• Genotoxin: a physical or chemical agent that can induce DNA damages
• DNA damage: is an abnormal chemical structure in DNA, which causes changes in the structure of genetic material and prevents the replication mechanism from functioning and performing properly
• Mutation: an alteration of the nucleotide sequence in the genome
• Mutagen: a physical or chemical agent that can induce mutations

Question 2

Genotoxin

Answer 2

• Mutagens: DNA adducts
• Clastogens: chromosome breaks
• Aneugens: mitotic spindle

Question 3

Carcinogenesis

Answer 3

• tumor formation is a complex multistep process

Depending on the mode of action, carcinogens induce or promote carcinogenesis
-> Genotoxic carcinogens: initiate tumor formation by DNA damage and
mutagenesis (e.g. by DNA adduct formation)
-> Non-genotoxic carcinogens: promote tumor formation by pathways other than DNA damage (e.g. cell proliferation, cytotoxicity, hormonal effects)

Question 4

Genotoxic carcinogens

Answer 4

• Direct DNA damage
• can be mutagenic
• Not concentration-dependent
• DNA damage is reversible; mutation not
  -> In theory: One DNA damage event can potentially lead to tumor formation

Human genotoxic carcinogens
- Aflatoxin B1: moldy food
- Aristolochic acid: traditional Chinese medicine
- benzo(alpha)pyrene: tobacco smoke
- formaldehyde: tobacco smoke
- nitrosamine: processed food, tobacco smoke
- vinyl chloride: PVC manufacture

Question 5

Non-genotoxic carcinogens

Answer 5

• No direct DNA damage
• Non-mutagenic
• Concentration-dependent
• Reversible
  -> Provides growth advantage over normal cells

Human non-genotoxic carcinogens
- Asbestos: Manufacture, fire protection
- Cyclosporine: immunosuppressant
- 1,2-Dichlorobenzene: Agrochemical production
- Dioxins: Herbicides
- Estradiol: Hormone therapy
- Ochratoxin A: moldy food
- Phenobarbital: seizure treatment

Question 6

Common types of DNA damages -> oxidative damages

Answer 6

• Life in oxygen-rich environment is a constant struggle to minimize oxidative damages
• 8-oxo-2’-deoxyguanosine (8-oxo-dG) is the most common oxidative lesion in duplex DNA
  -> guanine has a lower one-electron reduction potential than other nucleosides
• Human cells consist of highly efficient DNA repair pathways for oxidative damages -> Low mutagenic potential

Question 7

Natural endogenous DNA damages -> Abasic (AP) sites: Depurinations, depyrimidinations

Answer 7

• β-N-gycosidic bond between base and deoxyribose is hydrolyzed
• Depurination occurs at a higher frequency than depyrimidination (weaker basicity)

Question 8

Natural endogenous DNA damages -> Single-strand breaks (SSBs) are the most common lesions arising in cells

Answer 8

-> directly by disintegration of an oxidized sugar
-> indirectly during the DNA base-excision repair (BER)
* DNA double strand breaks are the most severe damages in the cell
* Repair of double-strand breaks can result in loss and rearrangement of genomic sequence

Question 9

Natural endogenous DNA damages -> DNA alkylation (O6-methylguanine)

Answer 9

• O6-MG (alkylation of the oxygen atom of guanine) is highly mutagenic
• 1 of 8 unrepaired O6-MG damages is leading to a mutation
• Repaired by O6-methylguanine-DNA methyltransferase (MGMT)
• MGMT is a suicide enzyme (1 MGMT protein can repair 1 damage)

Question 10

Natural endogenous DNA damages -> Deamination: Cytosine deamination

Answer 10

Spontaneous: hydrolysis reaction of cytosine into uracil, releasing ammonia

Why is thymine and not uracil found in DNA?
-> very frequent deamination conversions of C to U would not be caught by error- checking for non-DNA bases
-> would tremendously increase the mutation rate (C-G base-pairs would change to U-A)
-> uracil in DNA is quickly recognized and removed by multiple uracil DNA glycosylases

Question 11

Activation of genotoxic carcinogens

Answer 11

• Many genotoxins are not genotoxic by themselves, but can form genotoxic metabolites through cellular metabolism (e.g. by cytochrome P450 enzymes)

Direct genotoxic carcinogens
- active without metabolic activation

Indirect genotoxic carcinogens (Pro-carcinogens)
- carcinogens after metabolic activation
- often species-, sex- and organ-specific

• Ultimate carcinogens are often highly reactive
• Usually electrophiles (i.e., molecules deficient in electrons)
• Attack nucleophilic (electron-rich) groups in DNA, RNA and proteins

Question 12

DNA damage is not random

Answer 12

• Modifications of DNA can be at one or more DNA bases (or sites) and can also be formed with the DNA phosphate backbone
  -> Exocyclic: number is superscripted
  -> Endocyclic: number has normal size
• Genotoxic substances can have specific reaction patterns:
  -> Aristolochic acid: N6 of adenosine, N2 of guanosine
  -> Benz[a]pyrene: N2 of guanosine
  -> Formaldehyde: N6 of adenosine, N2 of guanosine, N4 of cytidine
• Efficiency of DNA repair mechanisms can vary between different DNA damage sites
  -> Guanosin-methylation: Repair efficiency for N7 methylation is higher than for O6-methylation

Question 13

How to detect DNA adducts

Answer 13

• 32P-Postlabeling
• Immunoassay
• GC/MS and LC/MS

Question 14

From DNA damage to mutation

Answer 14

• Cell division is required to fix a DNA damage into a mutation
• Once a DNA damage is fixed in a mutation, it can not be repaired
• Due to the structure transition mutations are more likely than transversion mutations
• Transition mutations are less likely to result in amino acid substitutions -> wobble base pair
• Silent mutation: TAT/TAC -> Tyrosine, TAG/TAA -> Stop codon, AGC/AGT -> Serine, AGG/AGA -> Arginine

Question 15

Mutational signatures

Answer 15

Chemical exposures and DNA repair defects can lead to specific mutational patterns: Tobacco smoke, Temozolomide, Aristolochic acid, Aflatoxin B1, Defects in Mismatch repair

Question 16

Our protection against genetic damage

Answer 16

• Although tens of thousands of DNA damages occur per day per cell, tumor formation is rare event due to:
  -> Efficient DNA repair systems
  -> Many mutations are “silent”
  DNA mutations does not result in a change to the amino acid sequence
  -> Mutations in non-coding sequences (e.g. introns)
  -> Mutations in the third base of some codons
  (e.g.: Proline: CCT, CCC, CCA, CCG)
  (e.g.: Arginine: CGT, CGC, CGA, CGG, AGA, AGG)
  Exception: change in codon usage can result in change of translation rate
  -> Possibility of neutral mutations
  Amino acid exchange does not lead to an altered protein function
  e.g. exchange with a chemically similar amino acid (leucine/isoleucine)
  -> Most of our genes are not involved in cancer Mutations in these genes do not lead to cancer

Question 17

Tumor Initiation vs Promotion

Answer 17

DNA damage is prerequisite, but not sufficient for carcinogenesis

Question 18

Genotoxic vs non-genotoxic carcinogens

Answer 18

Genotoxic mechanisms:
- DNA adducts
- Chromosome breakage, fusion, deletion, mis-segregation- non disjunction
-> leads to Genomic damage

Non-genotoxic mechanisms:
- inflammation
- immunosuppression
- reactive oxygen species
- receptor activation
- epigenetic silencing
-> leads to altered signal transduction

Question 19

Non-genotoxic carcinogens

Answer 19

Non-genotoxic carcinogens lead to increase in cell proliferation (inhibition of apoptosis)
-> Direct mitogens (mimic growth factors)
-> Cytotoxic compounds (regenerative cell proliferation)
-> Hormone or hormone mimetics (high concentrations or with similar receptor affinity)
-> Compounds affecting the immune system (pro- and anti-inflammatory factors)

Can non-genotoxic carcinogen exposure lead to cancer without genotoxic carcinogen exposure?
“Bad luck”- mutations:
- Mutations that occur during cell replication without the contribution of genotoxic carcinogens
- Fast dividing cells undergo proliferation pressure and therefore have less time to repair DNA damages
- Non-genotoxic carcinogens can induce proliferation pressure
- Consequence: accumulation of “Bad luck” - mutations

Question 20

Identifications of chemical carcinogens

Answer 20

• e.g. as part of the drug safety testing procedure:
  Target discovery (100 projects) -> preclinic (20 compounds) -> Phase I (10 compounds) -> Phase II (5 compounds) -> Phase III (2 compounds) -> FDA approval and phase 4 (1-2 compounds) -> Commercial (1 compound)
• Duration : 8-10 years
• 99 % of potential drugs do not pass toxicity or clinical phase tests
• 4/5 fail in preclinical studies
• Total costs per FDA approved drug: 500-600 Million US$
• Approximately 2000 animals per approved drug

Question 21

Definition REACH

Answer 21

Registration, Evaluation, Authorisation and Restriction of Chemicals in the EU -> data on the hazardous properties of all substances manufactured or imported into the EU in quantities above 1 tonne per year (ca. 100.000 chemicals in europe)

Question 22

Safety testing (e.g. for drugs)

Answer 22

• carcinogenic properties are evaluated in mutagenicity and carcinogenicity studies
• Carcinogenicity studies are the most time- and cost-consuming studies (animal studies)
• acute toxicity (weeks)
• Pharmacokinetics (weeks to months)
• Mutagenicity (Genotoxicity) (months)
• reproduction toxicity ( months to years)
• chronic toxicity (years)
• carcinogenicity (years)

Question 23

Regulation of carcinogens

Answer 23

• Principle established by Paracelsus: any poison can be non-toxic if the dose is below an appropriate threshold
• But: no threshold dose at any exposure level for genotoxic carcinogens
• Pharmaceutical industry: drug development is stopped when genotoxicity tests are positive (exception: cancer therapy)
• For non-genotoxic carcinogens: may be used, if the intake level is below a threshold level, evaluated in acute toxicity studies

Question 24

Carcinogenicity testing

Answer 24

2-years Rodent Bioassay:
* Rats (Sprague Dawley or Wistar) and mice (B6C3F1)
* Males and Females
* One control and three dosis (MTD (Maximum tolerated does), MTD/2, MTD/4) -> appropriate dose level selection is critical
* 50 animals per group -> 400-600 animals / study
* Histopathological examination of 24 potential target organs for neoplastic lesions
* Selected route of administration in animals should mimic as closely as possible the intended or expected route of human exposure
-> There is no in vitro method available which is considered sufficient to serve as replacement for animal studies!

Question 25

Safety testing -> Genotoxicity

Answer 25

The standard test battery comprises several in vitro and in vivo tests: * Genotoxicity is generally assessed by detection of mutations, chromosome abnormalities or DNA breaks Ames test (in vitro) -> S.typhimurium and E.coli -> Selection of revertants -> Bacteria - gene mutations MLA/HPRT -> Selection of gene mutations -> Rodent or human cells - genes, chromosomes, nuclei Micronucleus test (in vitro or in vivo) -> Nucleus aberrations, Changes in structure or number of chromosomes -> Rodent or human cells - genes, chromosomes, nuclei Chromosomal aberrations -> Structure of chromosomes -> Rodent or human cells - genes, chromosomes, nuclei Comet assay (in vivo rather than in vitro) -> DNA strand breaks -> Rodent or human cells - Chromosomes * Any confirmatory in vivo follow-up test needs to cover the same endpoint as the one which showed positive results in vitro

Question 26

Bacterial mutagenicity testing: Ames test

Answer 26

* Based on bacterial tester strains (Salmonella typhimurium or Escherichia coli) auxotrophic for an essential amino acid (e.g. histidine) due to a mutation in the corresponding gene * Incubation of tester strains with a potential genotoxin (mutagen) * Induced mutations lead to increase in number of revertants, which are able to grow on minimal essential amino acid medium S9 rat liver fraction: * Bacteria do not possess mammalian metabolic enzymes * Supernatant from rat liver homogenate, centrifuged with 9000 x g for 20 min * Cytosol and microsomes * Enriched in metabolic enzyme activity (especially CYPs) * Cheap and fast: the Ames test is performed on a regular basis before any other test

Question 27

Mammalian mutagenicity testing: MLA TK test

Answer 27

In vitro gene mutation assays: - Thymidine kinase (TK) mouse lymphoma assay (MLA) * Nucleoside analogues are incorporated into DNA in TK-positive cells -> cytotoxic * Genotoxin-induced mutations in TK lead to resistance -> survival * TK: encoded at chromosome 11; detect mutagenic and clastogenic events * HPRT: Hypoxanthine phosphoribosyl transferase (HPRT) assay; HPRT is encoded at the X chromosome; large deletions in the X chromosome are lethal -> point mutations and exon deletions are detected -> lower spontaneous mutation frequency

Question 28

Genotoxicity testing: Micronucleus test

Answer 28

- clastogenic and aneugenic chemicals - in vitro and in vivo, mainly with mammalian lymphocytes - cytokines blocking using cytochalasin B (inhibits actin polymerization), binucleate cells - staining using DNA specific dyes or fluorescent labelled DNA probes

Question 29

Genotoxicity testing: Chromosome aberrartions

Answer 29

* Frequency of numerical and structural aberrations are recorded by the analysis of structural abnormalities * After predetermined intervals of exposure to the test substance, cells are treated with a metaphase-arresting substance (e.g. colcemide, colchicine) * Chromosome preparation involves hypotonic treatment of the cells, fixation and staining

Question 30

Genotoxicity testing: Comet assay

Answer 30

* Exposed single cells are seeded into an aqueous gel matrix and a direct current electric field is established to induce DNA migration towards the anode * DNA fragments induced by DNA strand breaks migrate faster than whole chromosomes * Undamaged DNA strands are too large and do not leave the cavity * Usually detects single strand breaks and double strand breaks * Alkaline conditions: AP sites and excision repair Limitations: * Very sensitive method -> standardized methodology * Also necrosis or apoptosis can induce DNA damage -> High concentrations give often positive results -> Genotoxic mechanism should be verified also with another method Cells embedded in agarose on microscope slide -> Lysis -> Alkaline incubation -> Electrophoresis -> Nucleiod without strand breaks or Nucleoid containing strand breaks -> Neutralisation -> Stain and visualization

Question 31

Regulation of carcinogens

Answer 31

* ALARA principle: As low as reasonably achievable MOE (Margin of exposure) = TD10 (Dose to give tumors to 10 % rats or mice) (mg/kg/day) EHE (Estimated human exposure) (mg/kg/day) * MOE > 10,000 EFSA (Europäische Behörde für Lebensmittelsicherheit) estimates the existing carcinogenic risk to be low * The more the MOE falls below 10.000, the higher the risk and the more urgent the need to minimize potential exposure * Why 10,000? -> consideration of uncertainty factors x 10 inter-species x 100 intra-species (humans) x 10 additional safety factor