Cancer: DNA Damage and Repair

Question 1

What molecules are affected by base dimers and chemical cross-links?

Answer 1

DNA molecules. They are chemically linked up.

Question 2

What is damaged by base hydroxylations and how?

Answer 2

DNA bases can be oxidised, meaning the DNA has to be repaired and can become mutated during this repair process.

Question 3

Describe what is meant by abasic sites

Answer 3

During repair process, an entire DNA base has been removed.

The sugar-phosphate backbone is maintained.

This will cause problems during replication if unrectified.

Question 4

Describe the function of topoisomerase

Answer 4

It is a physiological enzyme involved in single strand breakages of DNA, allowing it to unwind during transcription/translation, which is subsequently reannealed.

Single strand breaks are not a problem.

Question 5

Describe why doubls strand breaks are so bad

Answer 5

There is a tendancy for the split molecule of DNA to drift apart. This is intolerable for the cell.

Repair mechanisms attempt to amend this but can fail and introduce DNA damage/mutation.

Question 6

Describe why DNA adduction and alkylation is bad

Answer 6

DNA polymerase cannot get past the bulky adduct and so cannot introduce the correct base.

Question 7

What kind of molecules for DNA adducts and alkyls?

Answer 7

Electrophiles

Question 8

Define insidious chemicals

Answer 8

They have an inherent property whereby the body can metabolise them into things that can damage DNA

Question 9

Give three phase 1 metabolism reaction examples

what is the common group introduced in these reactions?

what catalyses these reactions?

Answer 9

Oxidation, reduction, hydrolysis

hydroxyl group

CYP450

Question 10

Give 6 examples of Phase 2 reactions

Answer 10

Sulfation, glucuronidation, acetylation, methylation, amino acid and glutathione conjugation

(acidic, endogenous molecules)

Question 11

What is the purpose of phase 2 reactions

Answer 11

To make the molecules more polar for excretion

Question 12

Give an example of a group of insidious chemicals

name one

Answer 12

Polycyclic aromatic hydrocarbons

benzo[a]pyrene

Question 13

What do polycyclic aromatic hydrocarbons do to DNA?

Answer 13

They form adducts

Question 14

Where are PAHs found?

Answer 14

From combustion of fossil fuels or tobacco

Common environmental pollutants

Question 15

Describe the key features of two step epoxidation of B[a]P and why it is so dangerous

Answer 15

B[a]P is very lipophilic and contaminates many foods and is abundant in the air

CYP450 converts it into B[a]P-7,8-oxide (an epoxide) which is electrophilic

Epoxide hydroxylase cleaves it to form B[a]P-7,8-dihydrodiol which is not electrophilic

CYP450 converts this non-reactive molecule into B[a]P-7,8-dihydrodiol-9,10-oxide (another epoxide). This is extremely electrophilic and reactive, therefore forming adducts with DNA.

Question 16

Where are aflatoxins found?

Answer 16

Poorly stored grains and peanuts

produced by Aspergillus flavus

Question 17

Descrive the epoxidation of aflatoxin B₁

Answer 17

Carbon-carbon double bond on aflatoxin B1 is a substrate for CYP450

This reaction generates an epoxide (aflatoxin B₁,2,3-epoxide)

This epoxide is very reactive and can adduct iwth guanine in DNA

Question 18

What do aflatoxins tend to cause?

Answer 18

Primary hepatocellular carcinoma

This is because the liver contains a lot of CYP450

Question 19

Describe the metabolism of 2-naphthylamine

Answer 19

Amine group of 2-naphthylamine is converted to hydroxide-amine group by liver CYP450 1A2

The resulting N-hydroxy-2-naphthylamine is very toxic

The liver detoxifies this via glucuronyl transferase, adding a glucuronide

However, in urine, the pH is acidic, meaning the glucuronide breaks off, forming a positively charged nitrenium ion (electrophilic) and adducts to DNA

Question 20

What does 2-naphthylamine tend to cause?

Answer 20

Bladder carcinomas

Question 21

Where can 2-naphthylamine be found?

Answer 21

Poorly regulated dye-stuffs

Question 22

What damage does UV radiation cause?

Answer 22

Dimerisation of pyrimidines in DNA (forms a cyclobutane dimer)

this can lead to mutation

Question 23

What pathology does UV radiation tend to cause?

Answer 23

Skin cancer

Question 24

How does ionising radiation cause DNA damage?

Answer 24

It generates free radicals in the cell

These include super oxide radical and hyxroxyl radical which possess an unpaired electron, making them electrophilic

They form covalent bonds with DNA

This can cause double and single strand breaks

as well as apurinic and apyrimidinic sites

and base modifications

Question 25

What are apurinic/apyrimidinic sites?

Answer 25

Sugar-phosphate backbone is still present but the base has been removed.

Question 26

Give two examples of base modifications

Answer 26

Ring-opened guanine and adenine

Thymine and cytosine glycols

Question 27

What is p53’s role in modulating a cell’s response to stress?

what does MDM2 do?

Answer 27

When p53 is bound to MDM2 it is inactive

If the cell is exposed to stress, MDM2 releases p53

p53 molecules come together to form a tetramer and co-ordinate a response depending on levels of stress

i.e. mild/moderate stress = protection pathways; severe stress = senescence or apoptosis

Question 28

What does photolyase do?

Answer 28

Enzyme that splits cyclobutane pyrimidine-dimers

Question 29

Which two enzymes selectively remove methyl and alkyl groups from bases respectively?

Answer 29

Methyltransferases and alkyltransferases

Question 30

Which enzymes are involved in base excision repair?

What kind of damage does this repair?

Answer 30

DNA glycolases and apurinic/apyrimidinic endonucleases cut out affected area

repair polymerases (e.g. pol-beta) fills gap and DNA ligase completes repair.

Apurinic/apyrimidinic damage

Question 31

What is nucleotide excision repair?

What is it used to repair?

Answer 31

XP Proteins (Xeroderma pigmentosum) proteins assemble at site of damage and remove a long stretch of bases (normal and damaged)

helicase unwinds DNA, repair polymerases (e.g. pol-delta/beta) fill gap, and DNA ligase completes repair.

Mainly for bulky DNA adducts

Question 32

Describe the Ames test

Answer 32

Bacteria are genetically engineered to not produce histidine (required for growth).

Pharmaceutical chemical that needs to be tested is mixed with bacteria. If the bacterial DNA mutates, it will regain its ability to produce histidine, and the colony will grow.

Therefore if the bacterial grow, the chemical has mutagenic properties.

Question 33

How do you check whether chemicals cause chromosomal abberations?

Answer 33

In-vitro micronucleus assay

or bone marrow micronucleus assay in mice and rats

Question 34

Describe how in-vitro micronuclear assays can be used to detect chromosmal damage

Answer 34

Cells are treated with the chemical to be tested

Cytokinesis is blocked using cytochalasin-B

Binucleate cells are assessed for presence of micronuclei (form off fragments of chromosomes next to daughter nuclei)

Kinetochore proteins can be stained to determine wheter the chemical caused clastgenicity (chromsomal breakage) or aneuploidy (chromosomal loss)

Question 35

Describe bone marrow micronucleus assay in mice and rats

Answer 35

Pluripotent nature of bone marrow producing RBCs allows you to determine whether the chromosomes of newly formed cells can be damaged by the chemical you want to test

RBCs usually lose their nucleus but cannot remove small fragments of DNA

Presence of micronuclei indicates DNA damage