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Molecular basis of carcinogenesis I - Coggle Diagram

- - - - Control cell proliferation + apoptosis
- - - - Base-excision repair
  - - - Nucleotide-excision repair
  - - - Homologous recombination repair (HRR), non-homologous end joining (NHEJ)
  - - - Mismatch repair
- - - - Formation of oncogenic fusion proteins
- - - - Chemicals with positively charged moeity (electronegativity)
      - React with electron-rich sites (DNA adducts)
      - Free/lone electron pairs on DNA bases react with electrophilic chemicals
      - Examples
        
        Epoxides
        
        Lactones
        
        Alkyl sulphonates
        
        Platinum compounds
        
        Some nitrosamines
        
        Nitrogen/sulphur mustards
        
        Larger alkylation adducts can block DNA replication
        
        Due to size or crosslink formation
        
        Toxicity mediated by formation of +ve charged sulphonium ion + subsequent reaction with DNA (monoadducts + crosslinks)
        
        Alkylating agents
        
        Small DNA methylations + ethylations
        
        Modify nucleotides + alter base-pairing properties
      - Crosslinks vs monoadducts
        
        86% sulphur mustard lesions are monoadducts
        
        Sulphur mustard approx. 100-fold more toxic than monofunctional analogue
        
        Toxicity of DNA crosslinks
        
        Crosslinks cause DS breaks
        
        Replication fork stalling + processing of crosslink
        
        High toxicity during replication
        
        Ionising radiation can directly cause DS breaks
      - Cisplatin
        
        Aquation causes +ve charge intermediate
        
        Positive charge = site of nucleophilic attack
        
        e.g. by free electron pairs in guanine
    - - Polycytic, aromatic and planar molecules
      - Insert between base pairs in DS DNA
      - Distort normal spacing beween base pairs - insertions/deletions
      - e.g.
        
        Ethidium bromide
        
        Doxorubicin
    - - Hydrogen peroxide + superoxide naturally produced during respiration
        
        Leads to production of hydroxyl radicals
        
        Fenton reaction
      - Cells have mechanims to remove superoxide + hydrogen peroxide
        
        Superoxide dimutase
        
        Catalase
      - Effects on DNA
        
        8-oxodeoxyguanosine/8-hydroxydeoxyguanosine (8-OdG/8-OHdG)
        
        8-OHdG is pro-mutagenic
        
        Mis-incorporation of adenine during DNA replication
        
        Biomarker for DNA damage
        
        Excreted in urine
        
        Damaged base is excised by OGG1
        
        Part of excicison-repair pathway
        
        Detected and quantified by LC-MS
        
        Low in high antioxidant diets
    - - UV-radiation
      - Sources
        
        Natural
        
        Cosmic rays from the sun + outer space
        
        Radtioactive elements in soil, water, stone + atmosphere (radon gas)
        
        Artifical sources
        
        Diagnostic X-rays
        
        Nuclear testing
        
        Nuclear power plants
        
        Various products, smoke detectors, airport x-rays etc.
      - Ionising radiation
        
        Direct damage
        
        DS + SS breaks
        
        Indirect damage
        
        Free radicals + ROS produced
        
        DS + SS breaks
        
        Posesses enough energy to liberate an electrons from atoms
        
        DS breaks are highly toxic and mutagenic
  - - - PAHs
      - Aromatic amines
      - Some nitrosamines
      - Aflatoxin B
      - P450s
        
        Involved inactivation of >90% of pro-carcinogens
      - Additional role with for epoxide hydrolase
      - Phase 2 enzymes also involved in activation reations
    - - Benzo[a]pyrene diol epoxide (BPDE) produced (carcinogen)
- - - - Cytosine, uracil
- - - - Activate PKC
        
        Activation of signalling pathways (proliferation/divison)
    - - Activates aryl hydrocarbon receptor (AhR)
        
        Increased expression of P450 metabolic enzymes
        
        Increased proliferation e.g. Ras
        
        Decreased apoptosis
- - - - Micronucleus/metaphase spread test
        
        Cytokinesis-block micronucleus (CBMN) assay
        
        Micronuclei are whole chromosomes or fragments of chromsomes
        
        Can be caused by DS breaks or failure to attach to mitotic spindle
        
        In vitro
        
        Expose to test compound
        
        Block cytokinesis (cytochalasin B)
        
        Look for micronuclei in binucleated cells i.e. viable cells
        
        Can detect chromosome fragments + chromosomes that havent attached (clostogens and aneugens)
      - Mammalian cell mutation test
        
        HPRT gene mutation assay
        
        HPRT involved in nucleotide synthesis
        
        Expose to compound with and without S9 for 3-6hrs
        
        Culture in presence of 6-TG (toxic analogue of guanine)
        
        Only HPRT mutants grow
        
        Count colonies formed over 7-14 days
        
        Forward mutation assay
        
        Requires mutation of wt gene for cell growth
        
        Positive controls
        
        3-methylcolanthrene (+S9)
        
        Ethylnitrosourea (-S9)
        
        Same principle of mouse lymphoma assay (MLA)
      - Bacterial Ames assay
        
        Bacterial reverse mutation assay
        
        Mutation of histidine synthesis genes
        
        Bacteria cannot grow in histidine-free growth medium
        
        Mutagens can 'reverse' mutation, allowing bacteria to grow
        
        Assay performed with and without S9 fraction
        
        S9 fractionated from liver homogenate
        
        S9 is important for metabolism of potential carcinogens into active form
        
        Shows if carcinogen needs metabolic activation or not
        
        If chemical being tested allows growth of histidine-free medium
        
        It suggests the chemical has cause reverse mutation of the histidine synthesis genes
        
        WHO
        
        Recommends the use of 5 strains
        
        Allows mutagens with different mechanisms of mutagenesis to be identified
        
        Disadvantages
        
        False positives
        
        Nitorglycerine isnt carcinogenic or mutagenic
        
        Release of nitric oxide by bacterial enzymes
        
        NO can be mutagenic
        
        False negatives
        
        Only 70% of human carcinogens show as positive in Ames test
    - - Cytogenetic test
      - Gene mutation test
      - Transgenic animal mutation test
        
        Mouse models
        
        Bacteriophage shuttle vector used to introduce LacZ/LacI into mouse genome
        
        Treat with test compound, isolate DNA from mouse + perform reporter assay to assess mutation of bacterial gene
        
        'Big Blue Mouse' and 'MutaMouse'
        
        Mice are genetically engineered to have
        
        Phage packaging DNA
        
        Part of LacZ gene as reporter
        
        RE sites to allow isolation of reporter DNA insert from mouse genome
      - Used if in vitro tests are positive
    - - Used if intial in vitro testing is equivacal or contradictary
      - Comet assay
        
        Single cell gel electrophoresis assay
        
        In vitro or in vivo
        
        Lymphocytes from mice
        
        Embed cells in agarose on microscope slide, lyse + denature DNA (strong alkali)
        
        Electrophoresis, DNA stain, fluorescence microscopy, image analysis
        
        Damaged DNA (strand breaks/fragments) migrates through gel
        
        Alkali method measures DNA strand breaks and 'alkali labile sites' (abasic sites converted to SS break after alkali lysis)
        
        Neutral method detects DS breaks
        
        Can be modified to detect crosslinks
        
        Use of specific enzymes (e.g. OGG1) after lysis converts oxidative adducts to strand breaks
        
        Analyse at least 100 cells per microscope slide
        
        Tail length in image (distance from comet head to end of tail) indicates extent of DNA damage
        
        Tail intensity (% of DNA in tail) indicates amount of damaged DNA
        
        Tail moment (tail length x (%tail DNA/100))
      - Transgenic animal mutation test
- - - - 70 known carcinogens
      - Inc carcinogens + co-carcinogens )e.g. benzo[a]pyrene + catechol)
        
        BP is metabolised to carcinogenic BPDE
        
        Causes distinct p53 mutations
        
        Codons 157, 248 + 273 are 'hot spots'
        
        Same mutations observed in vitro + in smoking-induced lung cancers
  - - - 1000s subsequently affected by cancer + birth defects
    - - Isotypes inc. Iodine-131, Caesium-137 + Strontium-90
        
        \(^{131}\)I T\(_\frac{1}{2}\) = 8 days
        
        \(^{137}\)CS and \(^{90}\)S T\(_\frac{1}{2}\) = ~30yrs
  - - - Increased oxidative stress
        
        Altered signalling + possible DNA damage
      - Long fibres interfere with mitotic spindle
  - - - Carcinogenicity of TCDD confirmed in experimental studies
      - Mechanism of TCDD
        
        Strong agonist of AhR
        
        Activates AhR-mediated txn (pathways involved in tumour promotion)
        
        Diversity if gene expression changes shown in microarray studies
        
        AhR KO mice resistant to TCDD toxicity
    - - Chloracne