Cancer 6: DNA damage & Repair Flashcards
What can damage the DNA?
Chemicals (carcinogens)
Radiation
DNA damage can lead to mutation –> may lead to cancer
What are the different types of DNA damage by carcinogens
- DNA adducts & alkylation
- Base hydroxylations & abasic sites formed
- Base dimers & chemical cross-links
- Double & single strand breaks (double strand breaks very damaging)
What are the two phases of metabolism?
Phase I
- addition of functional groups
e. g. oxidations, reductions, hydrolysis - mainly cytochrome p450-mediated
Phase II
- conjugation of Phase I functional groups
e. g. sulphation, glucuronidation, acetylation, methylation, amino acid and glutathione conjugation - Generates polar (water soluble) metabolites.
What are polycyclic aromatic hyrdocarbons?
Common environmental pollutants
Formed from combustion of fossil fuels
Formed from combustion of tobacco
What does the two step epoxidation of B[a]P cause?
See slides
Benzo[a]pyrene –> converted to benzo[a]pyrene-7,8-oxide with P450
Epoxide hydroxylase converts to another compound then it is converted again by p450 to a group with a positive charge
This molecule DNA adducts
Epoxides are very reactive –> generating positive charge carbon atoms.
Describe the epoxidation of aflatoxin B1?
- Formed by Aspergillus flavus mould
- Common on poorly stored grains and peanuts
- Aflatoxin B1 is a potent human liver carcinogen, especially in Africa and Far-East
Aflatoxin B1 converted to aflatoxin B1,2,3-epoxide by P450
Describe the metabolism of 2-naphthylamine?
Past components of dye-stuffs
Include 2-naphthylamine and benzidine (potent carcinogen)
Potent human bladder carcinogens - DNA reactive electrophile
P450 produces a toxic product but in the liver the glucuronyl transferase removes this toxicity by adding a glucuronide group. However when it is excreted in the urine the pH means it gains a Nitrenium ion –> electrophile
What does solar (UV) radiation do?
Forms pyrimidine (thymine) dimers Skin cancer
What does ionising radiation do?
- Generates free radicals in cells
- Includes oxygen free radicals
1) super oxide radical: O2•
2) hydroxyl radical: HO• - These possess unpaired electrons
1) electrophilic and therefore seek out electron-rich DNA
Describe oxygen free radical attack on DNA??
Double and single strand breaks. Double strand breaks more damaging.
Apurinic & apyrimidinic sites
Base modification
- ring-opened guanine & adenine
- thymine & cytosine glycols (two hydroxys on the molecules)
- 8-hydroxyadenine & 8-hydroxyguanine (mutagenic)
What are the different types of DNA repair?
Direct reversal of DNA damage
- photolyase splits cyclobutane pyrimidine-dimers
- methyltransferases and alkyltransferases remove alkyl groups from DNA bases
Base excision repair (mainly for apurinic/apyrimidinic damage - damage where there is a loss of base)
- DNA glycosylases and apurinic/apyrimidinic endonucleases + other enzyme partners
- A repair polymerase (e.g. Polb) fills the gap and DNA ligase completes the repair.
Nucleotide excision repair (mainly for bulky DNA adducts)
- Xeroderma pigmentosum proteins (XP proteins) assemble at the damage. A stretch of nucleotides either side of the damage are excised.
- Repair polymerases (e.g. Pold/b) fill the gap and DNA ligase completes the repair.
During- or post-replication repair
- mismatch repair
- recombinational repair
Describe the process of DNA excision repair?
Base excision repair pathway
1) Mutagen exposure
2) DNA-glycosylase
3) AP-endonuclease
4) DNA polymerase
5) DNA ligase
Nucleotide excision repair pathway
1) Mutagen exposure
2) Endonuclease (cuts large parts of DNA)
3) Helicase
4) DNA polymerase
5) DNA Ligase
See slides
Describe the rate of endogenous damage and repair?
The greater the persistence of damage then the greater the chance of a mutagenic event
The cell has good repair capability. It only struggles when you throw carcinogens at it.
Describe the different fates of carcinogen-DNA damage?
See slides
Carcinogen damage leading to altered DNA –>
1) Efficient repair
2) Normal cell
1) Apoptosis
2) Cell death
1) Incorrect repair/altered primary seqeuence
2) DNA replication and cell division: fixed mutations
3) Transcription/translation giving aberrant proteins
3) Carcinogenesis if critical targets are mutated: Oncogenes, tumour suppressor genes.
How do you test for DNA damage
See slides
1) Look at the structures
2) In vitro BACTERIAL gene mutation assay e.g. Ames test with S. typhimurium. See if the chemical can damage the bacterial DNA.
3) In vitro MAMMALIAN CELL assay
e.g. chromosome aberration,
TK mutation in mouse lymphoma cell
Micronucleus assay
4) In vivo MAMMALIAN assay
e.g. Bone marrow micronucleus test
transgenic rodent mutation assay
5) Investigative in vivo MAMMALIAN assays
