Cancer 6: DNA Damage + repair Flashcards
how can carcinogens cause DNA Damage?
- 4 main methods :
- DNA adducts + alkylation
- Base dimers + chemical cross-links
- Double + single strand breaks
- Base hydroxylations + abasic sites formedz
Describe the 2 step expoxidation of Benzo(a)Pyrene = B[a]P
Two-step oxidation of Benzo(a)Pyrene (B[a]P)
e.g Benzo(a)pyrene.
- P450 oxidises B[a]P –> generates new oxide functional group –> forms the toxic chemical benzo(a)pyrene-7,8-oxide (toxic)
- Epoxide Hydroxylase (EH) uses the new functional group, and breaks it up –> to form the non-toxic Benzo(a)purene-7,8-dihydrol
- BUT P450 then uses this metabolite to generate another more toxic epoxide –> benzo(a)pyrene-7,8-dihydrodiol-9,10-oxide
- This new compound has the ability to damage DNA.
- -> highly positively charged (reactive electrophile species)
- -> binds with electron rich DNA (Especially guanine) to form a DNA adduct
Describe the metabolism of 2-naphthylamine
-2-naphthylamine = potent human bladder carcinogen
Metabolism of 2-naphthylamin:
- CYP1A2 converts the amine –> hydroxylamine (toxic + damages DNA)
- A phase II enzyme (Glucuronyl Transferase) detoxifies the substrate by adding a sugar molecule to it.
BUT in the bladder: the new metabolite = labile in the acid pH of the urine.
–> which generates positively charged amino group that is a highly DNA-reactive electrophile.
• DNA is particularly electron-rich due to its nitrogenous bases. So the new
metabolite reacts with the DNA, forming DNA adducts.
How does Solar UV radiation act as a carcinogen?
UV –> can form Pyrimidine (thymine) dimers
- can cause Skin cancer
How does ionizing radiation act as a carcinogen?
ionizing radiation:
- Generates free radicals in cells
- Includes oxygen free radicals
- -> super oxide radical
- -> hydroxyl radical (more reactive)
- Possess unpaired electrons
- -> electrophilc –> so seeks out DNA (electron rich)
What happens in oxygen free radical attack on DNA?
- Double and single strand breaks
- Apurinic & apyrimidinic sites –> body recognizes damaged bases –> wrong bases placed in
- Base modifications
–> ring opened guanine + adenine
–> tymine + cytosine
glycol
–> 8-hydroxyadenine & 8-hydroxyguanine = not recognized as proper base –> so has to guess complementary base
(mutagenic = prone to be changed during replication)
What enzyme system is most frequently involved in the activation of chemicals to metabolites that can damage DNA?
a. Glucuronyl transferase
b. Haem oxygenase
c. Cytochrome P450
d. Xanthine oxidase
e. Glutathione transferase
c. Cytochrome P450
What is the role of p53 in dealing with cellular stress?
- mild stress = regulates P53 target genes
- severe stress = apoptosis
What are the 4 main types of DNA repair?
- Direct reversal of DNA damage
- -> proteins sense problem –> reverses it
- -> eg photolyase
- -> e.g methy/alkyltransferases –> removes alkyl group from bases - Base excision repair
- -> usually for apyramidine / apurinic damage
- -> e.g DNA glycslases –> repaires DNA bases that have been removed
- -> polymerase =–> identifies gap in DNA –> and fills it in - Nucleotide excision repair
–> mining for bulky DNA adducts
–>
repaire enzymes recognise bulky adduct –> attempts to repair
–> e.g XP proteins assemble
repair polymerase –> fills gap + DNA ligase completes repair - During- or post-replication repair
- -> during = mistakes = identified + corrected
e. g mismatch repair
- -> e.g recombination repair –>
The greater the persistence of damage then the greater /lesser the chance of a mutagenic event
The greater the persistence of damage then the greater the chance of a mutagenic event
Fate of carcinogen-DNA damage
lead to altered DNA
a) if can be repaired
- -> cell remains normal
b) if cell = overwhelmed with DNA damage
- -> apoptosis occurs
c) if some cells = incorrectly repaired –> causes mutation
- -> causes carcinogens if critical targets are mutated OR abberant proteins are formed via transcription / translation
What are the different testings for DNA damage?
a) In vitro BACTERIAL gene mutation assay (Bacteria)
b) In vitro MAMMALIAN CELL assay (cell culture)
c) In vivo MAMMALIAN assay (rats)
d) Investigative in vivo MAMMALIAN assays (humans)
Describe the Bacterial (Ames) test for mutagenicity of chemicals
- grow bacteria on chemical to be tested
- give metabolic capability –> rat liver enzyme preparation
- give bacterial strain that cannot synthesize histidine
e. g salmonella - colonies form
- no. of colonies forming is proportional to extent of DNA damage chemicals has on cells
How would you detect DNA damage in mammalian cells - chromosomal abberations
- Treat mammalian cells with chemical in presence of liver S9.
- Look for chromosomal damage
Describe how in vitro micronucleus assay can be used to test for DNA damage?
- Cells = treated with chemical and allowed to divide
- Binucleate cells = assessed for presence of micronuclei
- micronuclei = indication of DNA damage
note: Can stain the kinetochore proteins to determine if chemical treatment caused clastgenicity (chromosomal breakage) or aneuploidy (chromosomal loss)
Which of the following is involved in the repair of damaged DNA?
a) Mutation
b) Epoxidation
c) DNA adduction
d) Base excision repair
e) Sister chromatid exchange
d) Base excision repair
What can damage DNA?
- chemicals:
- dietary
- lifestyle (e.g smoking)
- environmental
- occupational (e.g laying asphalt/coal works)
- medical
- endogenous - Radiation
- ionizing
- solar
- cosmic
what are the 2 types of mammalian metabolism?
phase 1 –> addition of functional groups
phase 2 –> conjugation of phase 1 functional groups
–> generates polar metabolites
how are Polycyclic aromatic hydrocarbons formed?
- wood burning / see smoke –> generates polycyclic aromatic hydrocarbons
- environmental pollutant
- formed from tobacco combustion
- formed from fossil fuel combustion
- aromatic benze ring fused together –> electron rich
Epoxidation of aflatoxin B1
- formed by aspergillum flavus mould
- commonly found on peanuts
- potent human liver carcinogen esp in africa + far east
- P450 converts aflatoxin B1 –> Aflatoxin B1 2,3 epoxide
- -> then forms bulky chemical which can cause DNA damage
Describe the process of excision repair of DNA damage
describe both method A + B
2 strands of DNA –> exposed to mutagen
Method A: base excision repair pathway: - DNA Glycosylase cuts base + adduct from DNA strand --> forms gap --> endonuclease cut DNA strand DNA polymerase put in app base --> ligase joins it up
Method B: Nucleotide excision repair pathway:
- endonuclease chops either side of the problem in the DNA
- helicase unwinds DNA
- -> allows access tot he problematic stretch
- -> facilitates loss of damaged DNA
- DNA polymerase adds in app base
- -> DNA ligase
Describe how in vivo micronucleus assay can be used to test for DNA damage?
- treat animals with chemical + examine bone marrow cells OR peripheral erythrocytes for micronuclei
