DNA damage and repair Flashcards
What can damage DNA?
Chemicals (carcinogens):
- dietary
- lifestyle
- environmental
- occupational
- medical
- endogenous
Radiation:
- ionising
- solar
- cosmic
Why is DNA damage a problem?
DNA damage can lead to mutation.
Mutation may lead to cancer.
What types of damage can occur to DNA?
Base dimers and chemical cross links.
Base hydroxylations and abasic sites formed.
Double and single strand breaks.
DNA adducts and alkylation.
What happens in Phase I metabolism?
Addition of functional groups, e.g. oxidations, reductions, hydrolysis.
Mainly cytochrome P450 mediated.
What happens in Phase II metabolism?
Conjugation of Phase I functional groups, e.g. sulphating, glucuronidation, acetylation, methylation, amino acid and glutathione conjugation. Generates polar (water soluble) metabolites.
What are polycyclic aromatic hydrocarbons?
Common environmental pollutants formed from combustion of fossil fuels and tobacco. Carcinogenic, damage DNA.
Describe the 2 step oxidation of benzo[a]pyrene (B[a]P) and why it is problematic.
B[a]P is a substrate for CYP450, which oxidises it to form an oxide (benzo[a]pyrene-7,8-oxide).
This oxide is a reactive electrophile.
There is a defence mechanism in the body- epoxide hydroxylate cleaves the 3-membered strained ring of the oxide to form a dihydrodiol (benzo[a]pyrene-7,8-dihydrodiol)- this is not toxic.
Dihydrodiol is also a substrate for P450, and is converted into another oxide (benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide).
This is a very reactive electrophile.
The best source of electrons is DNA, so DNA adducts are formed.
What is aflatoxin B1?
Formed by Aspergillus flavus mould.
Common on poorly stored grains and peanuts.
Potent human liver carcinogen, especially in Africa and Far-East.
Why is aflatoxin B1 carcinogenic?
Substrate for P450, epoxide added.
Reactive, unstable electrophile.
Reacts with N7 position on guanine, forms big chemical adduct on DNA.
DNA is damaged, fixed inappropriately, mutation.
What is 2-naphthylamine?
Past components of dye-stuffs include 2-naphthylamine and benzidine.
Potent human bladder carcinogens.
Why is 2-naphthylamine carcinogenic?
Amino group is a substrate for P450, converted to N-hydroxy derivative.
Glucuronyl transferase adds glucuronide to the molecule in the liver (phase II metabolism)- detoxified and excreted into urine.
Acid pH of urine hydrolytically strips off glucuronide (sugar).
DNA reactive electrophile produced (with nitrenium ion), causes bladder tumours.
Why is solar (UV) radiation carcinogenic?
Induces pyrimidine (thymine) dimers. Forms cross links. DNA replicating enzymes cannot work, cell tries to repair this and introduces mutation. Skin cancer.
Why is ionising radiation carcinogenic?
Generates free radicals in cells.
Includes oxygen free radicals.
Possess unpaired electrons- electrophilic and therefore seek out electron-rich DNA.
Give 2 oxygen free radicals.
Super oxide radical (O2*) Hydroxyl radical (HO*)
What types of DNA damage do oxygen free radicals generate?
Double and single strand breaks. Apurinic and apyrimidinic sites Base modifications: -ring-opened guanine and adenine -thymine and cytosine glycols -8-hydroxyadenine and 8-hydroxyguanine (mutagenic)
What enzyme system is most frequently involved in the activation of chemicals to metabolites that can damage DNA?
Cytochrome P450.
What is the role of p53 in dealing with cellular stress?
Tumour suppressor gene.
Usually with MDM2, which keeps p53 inactive.
When it is released from MDM2, it forms a dimer that activates many pathways to deal with mild, physiological or severe stress by repair or apoptosis.
What stresses can cause p53 to be released from MDM2?
Oxidative stress Hypoxia Nitric oxide Ribonucleotide depletion Mitotic apparatus dysfunction Oncogene activation DNA replication stress Double-strand breaks Telomere erosion
What are the actions of p53 when it is released from MDM2?
Regulation of p53 target genes:
- metabolic homeostasis
- antioxidant defence
- DNA repair
- growth arrest
- senescence
- apoptosis
Protein-protein interactions:
-apoptosis
What are the different types of DNA repair?
Direct reversal of DNA damage.
Base excision repair.
Nucleotide excision repair.
During- or post-replication repair.
What is involved in direct reversal of DNA damage for DNA repair?
Photolyase splits cyclobutane pyrimidine-dimers.
Methyltransferases and alkyltransferases remove alkyl groups from bases.
What is involved in base excision repair of DNA?
Mainly for apurinic/apyrimidinic damage.
DNA glycosylases and apurinic/apyrimidinic endonucleases and other enzyme partners.
A repair polymerase (e.g. Pol-beta) fills the gap and DNA ligase completes the repair.
What is involved in nucleotide excision repair of DNA?
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. Pol-delta/beta) fill the gap and DNA ligase completes the repair.
What is involved in during- or post-replication repair of DNA?
Mismatch repair.
Recombinational repair.
What happens if there is a small amount of carcinogen damage leading to altered DNA?
Efficient repair.
Normal cell.
What happens if there is excessive carcinogen damage leading to altered DNA?
Apoptosis.
Cell death.
What happens if the carcinogen damage is intermediate, leading to altered DNA?
Incorrect repair/altered primary sequence.
DNA replication and cell division- fixed mutations.
Transcription/translation giving aberrant proteins, or carcinogenesis if critical targets are mutated (oncogenes, tumour suppressor genes).
What is the tiered structure used to test for whether a drug or chemical causes DNA damage?
Structural alerts/SAR.
In vitro bacterial gene mutation assay, e.g. Ames test with S. typhimurium.
In vitro mammalian cell assay, e.g. chromosome aberration, TK mutation in mouse lymphoma cell, micronucleus assay.
In vivo mammalian assay, eg. bone marrow micronucleus test, transgenic rodent mutation assay.
Investigative in vivo mammalian assays.
What is the bacterial (Ames) test for mutagenicity of chemicals?
Chemical to be tested (with rat liver enzyme preparation, S9) is added to bacteria that do not synthesise histidine, e.g. Salmonella strain.
Conversion of chemical to reactive metabolite?
On histidine free media: if mutations occur in bacterial genome, bacteria acquire ability to synthesise histidine = colonies.
Quantitative test, answer within days.
How is chromosomal analysis used to detect DNA damage in mammalian cells?
Incubate cells with damaging chemical, in presence of liver S9. Karyotyping- has mammalian cell been damaged? -chromatid exchange -chromosome gap -double minutes -chromosome interchanges -acentric ring -chromosome break
Explain in vitro micronucleus assays used to detect DNA damage.
Cells treated with chemical and allowed to divide.
Binucleate cells assessed for presence of micronuclei.
Can stain the kinetochore proteins to determine if chemical treatment caused clastgenicity (chromosomal breakage) or aneuploidy (chromosomal loss).
Explain bone marrow micronucleus assays used in mice or rats to detect DNA damage.
Treat animals with chemical and examine bone marrow cells or peripheral blood erythrocytes for micronuclei.
