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Cancer > DNA Damage and Repair > Flashcards

DNA Damage and Repair Flashcards

1
Q

State some different types of DNA damage caused by carcinogens.

A
  1. Base dimers and chemical cross-links
  2. Base hydroxylations & abasic sites formed
  3. Double and Single strand breaks
  4. DNA adducts & alkylation
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2
Q

What are abasic sites?

A

During the repair process, the entire DNA base has been removed so the sugar backbone is maintained but we have removed the base from the mutagenic molecule
During replication, this missing base can cause problems

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3
Q

What are the implications of single strand breaks?

A

These are common and useful

Topoisomerase causes single strand breaks and it is involved in relaxing and unwinding the DNA before replication

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4
Q

What are the implications of double strand breaks?

A

These are NOT GOOD
The two strands have a tendency to drift apart when a double strand break occurs
There are repair mechanisms for dealing with this, but sometimes the DNA repair can go wrong and introduce DNA damage

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5
Q

What is the usual type of damage that is caused by chemicals?

A

DNA adducts

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6
Q

Why is DNA the target for many carcinogens?

A

Chemical carcinogens are usually metabolically activated and converted into electrophiles (they want electrons)
DNA is very electron rich

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7
Q

What are the consequences of bulky DNA adducts?

A

The electrophiles bind and form a covalent bond
The binding of these adducts causes problems, particularly during replication because it interferes with the ability of DNA polymerase to recognise the base (because of the bulky adduct)

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8
Q

What are the six types of Phase II reaction?

A 
Glucuronidation  
Acetylation  
Sulphation 
Methylation  
Amino acid conjugation  
Glutathione conjugation
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9
Q

What are polycyclic aromatic hydrocarbons? Give an example?

A

They are environmental pollutants formed from the combustion of fossil fuels and tobacco
- Benzo[a]pyrene

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10
Q

Describe two-step epoxidation of benzo[a]pyrene and how it can lead to DNA adducts

A
  1. B[a]P is a substrate for CYP450, which converts it to B[a]P-7,8-oxide (this is an electrophile) - v reactive
  2. The body has a defence mechanism – epoxide hydrolase converts the oxide to a dihydrodiol (B[a]P-7,8-dihydrodiol)
  3. This is inactive
    However, this dihydrodiol is also a substrate for CYP450, which converts it to another oxide (B[a]P-7,8-dihydrodiol-9,10-oxide)
  4. This even more reactive than the previous oxide – it goes on to form DNA adducts
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11
Q

State two past components of dyestuff that are potent bladder carcinogens.

A

Benzidine and 2-naphthylamine

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12
Q

Explain the mechanism by which 2-naphthylamine is a bladder carcinogen.

A
  1. the amine group of 2-naphthylamine is
    oxidised by CYP1A2
  2. Glucuronyl transferase adds a flucoriondie group to the amine which is then broken down down by the acidic urine pH.
  3. The nitrenium ion remaining is electrophilic so it leads to the formation of DNA adducts- bladder tumours
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13
Q

What does UV radiation lead to the formation of?

A

Pyrimidine (thymine) dimers – adjacent pyrimidines can covalently link - SKIN CANCER

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14
Q

What does ionising radiation generate?

A

Free radicals

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15
Q

Name 2 oxygen free radicals. What is a characteristic of them?

A 
Superoxide radical (O2.) 
Hydroxyl radical (HO.)
-
possess unpaired electrons
electrophilic and therefore seek out electron-rich DNA
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16
Q

What are the consequences of oxygen free radical attack on DNA?

A
  1. Single and Double strand breaks
  2. Apurinic and apyrimidic sites
  3. Base modifications:
    - Ring-opened guanine and adenine
    - Thymine and cytosine glycols
    - 8-hydroxyadenine and 8-hydroxyguanine
17
Q

What are the p53 mediated responses to mild and severe physiological stress?

A

Mild – repair the damage and restore the normal function of the cell
Severe – apoptosis

18
Q

What are the main types of DNA repair?

A
  1. Direct reversal of DNA damage
  2. Base excision repair
  3. Nucleotide excision repair
  4. During- and post-replication repair
19
Q

Give two examples of direct reversal of DNA damage.

A
  1. Photolyase looks for cyclobutane-pyrimidine dimers and cuts them
  2. Methyltransferases and alkyltransferases remove alkyl groups from the bases
20
Q

What comes under during or post replication repair?

A
  1. Mismatch repair

2. Recombinational repair

21
Q

Which base is most electron-rich and hence most capable of attracting electrophiles?

A

Guanine

22
Q

Describe the process of base excision repair. What is it mainly used for?

A
  • (mainly for apurinic/apyrimidinic damage)
    1. DNA glycosylase hydrolyses between the base and the sugar
    2. Then apurinic/apyrimidinic endonuclease splits the DNA strand so there is a gap in the backbone
    3. A repair polymerase (DNA polymerase beta ) then fills in the missing base (using the complementary strand as template)
    4. DNA ligase then seals the DNA
23
Q

Describe the process of nucleotide excision repair. What is it mainly used for? Give an example

A
  • mainly for bulky DNA adducts
    1. Xeroderma pigmentosum proteins (XP proteins) assemble at the damage. A stretch of nucleotides either side of the damage are excised.
  1. Repair polymerases (e.g. Pold/b) fill the gap and DNA ligase completes the repair.
24
Q

Describe the possible fates of carcinogen-DNA damage.

A

Carcinogen causing altered DNA –> incorrect repair/altered primary sequence –> DNA replication and cell division (fixed mutation) –>

a. transcription and translation giving aberrant proteins +
b. carcinogenesis if critical targets are mutated

25
Q

Describe the process of testing whether a chemical can cause carcinogenesis.

A

Look at structure of compound
Test in vitro on bacteria
Test in vitro on mammalian cells
Test in vivo on mammals

26
Q

Describe the bacterial (Ames) test for mutagenicity of chemicals.

A
  1. This test usually uses bacteria that do not produce histidine e.g Salmonella typhimurium
  2. The bacterium is genetically engineered so that it can’t produce histidine, so it can only survive and grow on a culture medium that has exogenous histidine
  3. The compound to be tested is, firstly, incubated with rat liver enzymes containing CYP450 enzymes to metabolise the chemical into an active form that can be carcinogenic
  4. The bacteria are mixed with the active chemical and then placed on a culture medium with NO histidine
  5. Any colonies that survive will have become mutated by the chemical so that it regains the ability to produce its own histidine and hence can grow in the absence of histidine
  6. Any bacteria that hasn’t been mutated will die on the dish
    The greater the DNA damaging capability of the chemical, the more colonies will grow in the absence of histidine
27
Q

Describe the use of in vitro micronucleus assays.

A

This is trying to measure the ability of a chemical to break up DNA into fragments

We need the cell to go through one replication cycle and then stop it when it’s at the binucleus stage – this is when you check for the presence of micronuclei

28
Q

What is used to block cytokinesis and hold the cell in the binucleate stage in the micronucleus assay?

A

Cytochalasin-B

29
Q

What are the two types of chromosomal damage that can be detected by this micronucleus assay?

A

Clastogenicity – chromosomal breakage

Aneuploidy – chromosomal loss/change in the number of chromosomes

30
Q

Explain the reasoning behind the use of bone marrow micronucleus assays to test the mutagenicity of a chemical.

A

Bone marrow is pluripotent

The animals are treated with the chemical and their bone marrow cells and peripheral erythrocytes are examined for the presence of micronuclei

Erythrocytes normally remove the nucleus during development, but it CANNOT remove small fragments of DNA e.g. a micronucleus
So the presence of micronuclei in erythrocytes indicates DNA damage

31
Q

What happens in Phase 1 metabolism? What group of enzymes mainly governs this?

A

addition of functional groups
e.g. oxidations, reductions, hydrolysis
mainly cytochrome p450-mediated

32
Q

What happens in Phase II metabolism?

A
  1. conjugation of Phase I functional groups
    e. g. sulphation, glucuronidation, acetylation, methylation, amino acid and glutathione conjugation
  2. Generates polar (water soluble) metabolites.
33
Q

What is aflatoxin B1? Where is it commonly found?

A

Formed by Aspergillus flavus mould

Common on poorly stored grains and peanuts

34
Q

In what organ is aflatoxin B1 mainly a carcinogen?

A

liver

35
Q

Outline the epoxidation of aflatoxin B1?

A
  1. P450 oxides aflatoxin B1
    Aflatoxin B1 then adducts to
  2. DNA directly using its adjacent N7 positively charged carbon atom
36
Q

How Ould you detect DNA damage in mammalian cells?

A

Treat mammalian cells with chemical in presence of liver S9. Look for chromosomal damage

Cancer (15 decks)

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