Cancer 6 - DNA damage and repair Flashcards
Chemicals and radiation can both cause cancer. Give an example of endogenous species damaging DNA
Mitochondria produce reactive oxygen species - these can damage DNA
6 ways in which carcinogens can damage DNA
- Base dimers and chemical cross links
- DNA Adducts and alkylation
- Base hydroxylations
- Abasic sites forming
- Single strand break (common and fixable)
- Double strand breaks (the worst thing)
There are 2 phases in mammalian metabolism. What happens in each phase
Phase 1 = addition of functional groups - cytochrome p450 mediates (via oxidation)
Phase 2 = conjugation of phase 1 functional groups - generates polar metabolites
POLAR = EASY TO EXCRETE
Name a common environmental pollutant found in smoke
Polycyclic aromatic hydrocarbons
Explain the 2 step oxidation of Benzo(a)pyrene (bap)
- bap oxidised by p450 to become bap-7,8-oxide
- Oxide is reactive and so epoxide hydroxylase cleaves 3 membered O ring
- This forms bap-7,8-dihydrodiol (not toxic)
- p450 converts dihydrodiol into another oxide (bap….dihydrodiol-9,10-oxide) - VERY REACTIVE
- It is an electrophilic species - DNA is a good source of electrons so DNA adducts are formed
Aflatoxin B1 can also be epoxidised. How
P450 converts aflatoxin b1 into aflatoxin b1-2,3-epoxide
This then targets guanine groups
Aflatoxin = potent LIVER carcinogen
2 naphthylamine was used in dyes. It targets which organ?
Bladder.
It includes benzidine also which is a carcinogen
How does 2-naphthylamine cause cancer
Generates nitrenium ion in the bladder due to urine ph which destabilises the glucuronide
- 2-naphthylamine converted to N-hydroxy-2-naphythylamine (toxic)
- Body defence (glucuronyl transferase) glucuronidates it. It becomes inert
- PROBLEM = urine pH generates a nitrenium ion from this - potent electrophile which can cause bladder tumours
Where do polycyclic aromatic hydrocarbons mainly cause cancer?
Various parts of the body
As p450 involved in its activation is found in several different tissues
Where do aflatoxins mainly cause cancer?
In the liver - as it is mainly activated by p450 enzymes found in the liver
What can solar (UV) radiation lead to?
Formation of pyrimidine dimers - covalent bond between both - can cause skin cancer
What can ionising radiation do?
Generate oxygen free radicals:
- Superoxide radical (O2’)
- Hydroxyl radical (OH’)
They are electrophilic so want to deposit their electron - DNA is v electron rich so DNA attacked
Which is more reactive, superoxide radical or hydroxyl radical?
Hydroxyl radical
What are 3 forms of oxygen free radical attack on DNA
- Double and single strand breaks (single = not so bad, double = much worse)
- Abasic sites (i.e. apurinic and apyrimidinic sites when base has been oxidised, so enzymes cut out the base but leave the sugar-phosphate backbone intact)
- Base modification
What are 3 forms of base modifications caused by oxygen free radical attack
- Ring opened guanine and adenine
- Thymine and cytosine glycols
- 8-hydroxyadenine & 8-hydroxyguanine (mutagenic)
Which enzyme system is most frequently involved in activation of chemicals to metabolites that can damage DNA?
Cytochrome p450
p53 is a crucial …..?
TSG - usually tied up w MDM2 which keeps it inactive
When activated by certain stimuli, it forms a dimer which activates different pathways
Under SEVERE stress, what does p53 activate?
Apoptotic pathway
What are the 4 types of DNA repair
- Direct reversal of DNA damage
- Base excision repair
- Nucleotide excision repair
- During-/post replication repair
Explain repair via direct reversal of DNA damage
- Photoylyase splits cyclobutane pyrimidine dimers (e.g. formed through solar radiation e.g.)
- Methyltransferases & alkyltransferases remove alkyl groups from bases
Explain briefly repair via base excision
(mainly for basic damage)
- DNA glycosylases & apurinic/apyrimidinic endonuclease involved
- Repair polymerases (e.g. Polbeta) fills gap and DNA ligase completes repair
Explain briefly repair via nucleotide excision
(mainly for bulky DNA adducts)
- Xeroderma pigmentosum proteins assemble at the damage (group of proteins). Stretch of nucleotides either side of damage are excised
- Repair polymerases (e.g. Poldelta) fill the gap - DNA ligase completes the repair
What is meant by during / post-replication repair
Refers to mismatch repair or recombinational repair
Which 2 pathways are the major ways in which DNA damage is repaired?
Base excision pathway and nucleotide excision pathway
What is the most electron rich base?
Guanine
Explain fully the base excision pathway
- DNA gycosylase splits bond between base and sugar
- AP endonuclease causes a gap in the sugar-phosphate backbone by splitting DNA strand
- DNA polymerase fills in missing base
- DNA ligase seals the DNA by adding a backbone to repair the DNA
Explain fully the Nucleotide excision pathway
- Endonuclease make 2 cuts, either side of the site of DNA damage
- Helicase removes this patch - leaving behind double stranded DNA with a patch missing
- DNA Polymerase replaces the bases
- DNA ligase seals the DNA to repair the DNA
Explain how it is determined if a chemical is carcinogenic (tiered approach)
- Structural alerts
- In vitro bacterial gene mutation assay
- Mammalian cell assay in Vitro
- In vivo mammalian assay - bone marrow micronucleus test
- Investigative in vivo mammalian assay
Give an example of in vitro bacterial gene mutation assay
e.g. Ames test for mutagenicity of chemicals
Explain the Ames test for mutagenicity
- Chemical “stew” incubated with rat liver enzyme (containing p450 to metabolise it)
- Bacteria that DOES NOT produce histidine used - typically salmonella typhimurium
- IF cell is mutated to be able to produce histidine, colonies start forming
- Number of colonies proportional to DNA damaging capability of the mutagen
Chromosomal aberrations are detected how?
Mammalian cells treated with chemicals in presence of liver S9. Look for chromosomal damage.
