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Pathomechanismen > Carcinogens > Flashcards

Carcinogens Flashcards

1
Q

Carcinogens

A
  • 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
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2
Q

Genotoxin

A
  • Mutagens: DNA adducts
  • Clastogens: chromosome breaks
  • Aneugens: mitotic spindle
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3
Q

Carcinogenesis

A
  • 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)

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4
Q

Genotoxic carcinogens

A
  • 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

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5
Q

Non-genotoxic carcinogens

A
  • 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

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6
Q

Common types of DNA damages -> oxidative damages

A
  • 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
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7
Q

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

A
  • β-N-gycosidic bond between base and deoxyribose is hydrolyzed
  • Depurination occurs at a higher frequency than depyrimidination (weaker basicity)
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8
Q

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

A

-> 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

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9
Q

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

A
  • 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)
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10
Q

Natural endogenous DNA damages -> Deamination: Cytosine deamination

A

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

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11
Q

Activation of genotoxic carcinogens

A
  • 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
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12
Q

DNA damage is not random

A
  • 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
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13
Q

How to detect DNA adducts

A
  • 32P-Postlabeling
  • Immunoassay
  • GC/MS and LC/MS
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14
Q

From DNA damage to mutation

A
  • 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
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15
Q

Mutational signatures

A

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

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16
Q

Our protection against genetic damage

A
  • 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
17
Q

Tumor Initiation vs Promotion

A

DNA damage is prerequisite, but not sufficient for carcinogenesis

18
Q

Genotoxic vs non-genotoxic carcinogens

A

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

19
Q

Non-genotoxic carcinogens

A

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

20
Q

Identifications of chemical carcinogens

A
  • 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
21
Q

Definition REACH

A

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)

22
Q

Safety testing (e.g. for drugs)

A
  • 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)
23
Q

Regulation of carcinogens

A
  • 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
24
Q

Carcinogenicity testing

A

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!

25
Q

Safety testing -> Genotoxicity

A

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
26
Q

Bacterial mutagenicity testing: Ames test

A
  • 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
27
Q

Mammalian mutagenicity testing: MLA TK test

A

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
28
Q

Genotoxicity testing: Micronucleus test

A
  • 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
29
Q

Genotoxicity testing: Chromosome aberrartions

A
  • 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
30
Q

Genotoxicity testing: Comet assay

A
  • 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

31
Q

Regulation of carcinogens

A
  • 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

Pathomechanismen (13 decks)

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