6. DNA Damage and Repair

Question 1

What can damage DNA?

Answer 1

• NOTE: most chemicals that we are exposed to must be metabolically converted (by us) to something that can damage DNA
• Diet is strongly associated with cancer (about 40-45% of human cancers)
• Herbicides and pesticides contribute to a relatively small proportion of the human cancer burden
• Medical treatments, such as radiotherapy, can also damage DNA and increase the risk of cancer
• Some things are endogenous, for example, mitochondria produce reactive oxygen species that have the ability to damage DNA
• As we grow older we accumulate more and more mutations, which could lead to a neoplastic phenotype

Question 2

What are the different forms of DNA damage by carcinogens. Describe them briefly.

Answer 2

• Base Dimer and chemical cross-links
  
  • ​This is where the DNA molecules are being chemically linked up
• Base Hydroxylations
  
  • ​An oxidative reaction occurring on one of the DNA bases which can cause problems
  • This could mean that the DNA has to get repaired and during the repair process, it could get mutated
• Abasic Sites
  
  • ​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, the missing base will cause problems
• Single Strand Breaks
  
  • ​These are very common and can be very useful
  • There are physiological enzymes that are responsible for making single strand breaks
  • Topoisomerase is involved in the relaxing and unwinding of DNA - it works by chopping the strand of DNA and allowing the strand to unwind and we can gain access to the DNA as the strand is re-annealed
  • So we can deal with single strand breaks in DNA.
• Double Strand Breaks
  
  • ​These are a bit of a disaster
  • After the double strand breaks, there is a tendency for the two bits of DNA to drift apart and this is intolerable from the cell’s point of view
  • There are a number of DNA repair mechanisms that attempt to amend this, but sometimes the DNA repair can go wrong and introduce DNA damage
• DNA Adducts and Alkylation
  
  • ​This is generally the type of damage that is caused by chemicals
  • Some chemicals ted to be metabolically activated into electrophiles (it really wants electrons)
  • DNA is very rich in electrons because of all the nitrogens in the bases
  • The electrophiles bind to the DNA and form a covalent bond
  • The binding of a big bulky chemical to the DNA causes problems particularly during replication because the DNA polymerase runs along the strand and wants to figure out which base to put in next, but it won’t be able to do this if it is bound to a big chemical group
  • In short, DNA polymerase cannot recognise the base because of the chemical adduct.

Question 3

Summarise the mammalian metabolism.

Answer 3

• In short:
  
  • Phase 1: introduce or unmask functional groups that can be used in Phase 2
  • Phase 2: we use the functional groups (made available by phase 1) to conjugate it with an endogenous molecule to make it water soluble so that it can be excreted in the urine
• Cyrochrome P450 enzymes (family of 57 enzymes) have a broad substrate specificity and is responsible for oxidising chemicals (very involved in Phase 1 metabolism)
• So the whole purpose of metabolism is to take something that is lipophilic and make it more polar so that we can get rid of it

Question 4

What are polycyclic Aromatic Hydrocarbons?

Answer 4

• YOU DO NOT NEED TO REMEMBER THE CHEMICAL STRUCTURES
• Polycyclic aromatic hydrocarbons are common environmental pollutants
• Whenever you see smoke, there are probably a lot of these polycyclic aromatic hydrocarbons in it
  *

Question 5

Explain the metabolism of Benzo[a]pyrene.

Answer 5

• This is a model compound that is used to study polycyclic aromatic hydrocarbons
• B[a]P is a substrate for CYP450, which oxidises it to form an oxide (Benzo[a]pyrene-7,8-oxide)
• This oxide is reactive and wants to find electrons (it is an electrophile)
• There is a defence mechanism in the body - epoxide hydroxylase cleaves the three membered strained ring of the oxide to form a dihydrodiol Benza[a]pyrene-7,8-dihydrodiol) - this is NOT TOXIC
• So far, we have converted something that is potentially toxic, to something that is toxic and then detoxified it
• THE PROCESS DOES NOT STOP HERE
• Unfortunately, the non-toxic dihydrodiol metabolite is also a substrate for P450
• So P450 converts this non-toxic metabolite into another oxide benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide
• This is very reactive (even more so than the previous reactive oxide) and is desperate to find some electrons to react with
• The best source of electrons is DNA so DNA adducts are formed

Question 6

Explain the metabolism of aflatoxin B₁.

Answer 6

• In the USA and western European countries the levels of aflatoxin are regulated whereas in eastern european countries and africa there is a higher prevalence of liver toxin
• Common in pore stored grains

Question 7

Describe the metabolism of 2-naphthylamine.

Answer 7

• 2-naphthylamine is a part component of dye-stuffs
• Benzidine is another important past component of dye-stuffs
• Both benzidine and 2-naphthylamine are potent BLADDER carcinogens
• Note: different targets of different carcinogens
  
  • Polycyclic Aromatic Hydrocarbons cause cancer in many different parts of the body because P450 is involved in its activation and is found in a lot of different tissues
  • Aflatoxins primarily target the liver because it is mainly activated by P450 that is found in the liver
• 2-naphthylamine is a substrate for CYP450, which converts the amino group to form a hydroxylamine (N-hydroxy-2-naphthylamine)
• Hydroxylamines are reactive
• In the liver, when this reactive hydroxylamine is formed, it is glucoronidated (detoxifying reaction) so the chemical is activated and then inactivated
  
  • The glucoronidation is done by glucuronyl transferase
• The inactive metabolite is excreted by the liver and it goes into the bladder and mixes with the urine
• Urine is ACIDIC, and, under acidic conditions, the glucuronides are hydrolysed
• This releases the hydroxylamine derivative, which in the acidic conditions, rearranges to form a positively charged nitrogen (nitrenium ion)
• The nitrenium ion is an electrophile which then goes and binds to the DNA and forms adducts
• The bladder isn’t as capable of detoxifying the hydroxylamine derivative as the liver

Question 8

Explain the effect of Solar (UV) radiation on DNA.

Answer 8

• UV radiation can lead to the formation of pyrimidine dimers –> strongly associated with skin cancers
• NOTE:
  
  • Pyrimidines = cytosine + uracil + thymine
  • Purines = adenine + guanine
• If there are 2 pyrmidines next to each other, under the presence of UV radiation, they can covalently link due to certain types of UV radiation
• Sun rays are quite rich in the damaging UV rays
• The main type of cancer that it causes is skin cancer

Question 9

Describe the types of ionising radiation and the free radicals they produce.

Answer 9

• Examples of ionising radiation: gamma, X-ray, beta particles.
• These all have the ability to generate chemistry within a cell
• They can generate free radicals
• Oxygen free radicals are produced by normal biochemistry (e.g. mictochondria produce oxygen free radicals) and there are good defence mechanisms for dealing with them - however ionising radiation can overwhelm the defence mechanisms
• Super Oxide Radical - very powerful - this is a molecule oxygen that has an extra electron so it is very reactive
• NOTE: If there is an extra electron in the outer orbit, then the molecule will be very reactive
• Hydroxyl Radical - a hydroxyl group that has grabbed an extra electron - this is even more reactive than the super oxide radical
• These free radicals are very electrophilic and DNA is electron rich

Question 10

Describe the oxygen free-radical attack on DNA.

Answer 10

• Single stranded breaks - not a big deal we can sort these out
• Double strand breaks - very damaging
• Apurinic and apyrimidic sites - base has been oxidised by an oxygen free radical and the DNA repair enzymes come and cut out the base itself, leaving the sugar-phosphate backbone in atct so there are gaps (abasic sites)
• You can also get base modifications:
  
  • ​Ring-opened guanine + adenine
  • Thymine + cytosine glycols
  • 8-hydroxyadenine + 8-hydroxyguanine (particularly mutagenic - prone to change during replication)

Question 11

Review the role of p53 in dealing with cellular stress.

Answer 11

• p53 has a very important role - it is a crucial tumour suppressor gene
• It is normally tied up with MDM2, which keeps p53 inactive
• When it is released from MDM2, it forms a dimer that activates many pathways
• If we have mild physiological stress e.g. DNA repair or growth arrest, p53 orchestrates a transcriptional series of events and activavtes proteins that help repair the problem
• If there is severe stress, the p53 can activate an apoptotic pathway by directly interacting with apoptosis proteins
• Many different stresses can kick p53 into action

Question 12

Name and describe the types of DNA repair.

Answer 12

• Direct reversal of DNA damage
  
  • ​Photolyase looks specifically for cyclobutane-pyrimidine dimers and cuts them
  • NOTE: solar radiation generates these dimers in the first place
  • These photolyase enzymes cut out the dimers and restore the normal sequence
  • Methyltransferases and alkyltransferases - remove alkyl groups from bases
  • REMEMBER: methylation and demethylation is an important way of controlling gene expression
  • Sometimes, you can get inappropriate groups to restore the DNA structure
• Base excision repair - mainly for apurinic/apyrimidic damage
  
  • This is damage where the base has been removed from the DNA
  • DNA glycosylases and apurinic/apyramidic endonucleases are interested in repairing this damage
  • Repair polymerase fill the gap caused by the missing base and DNA ligase repairs it
• Nucleotide Excision Repair - mainly for bulky DNA adducts
  
  • E.g. if polycyclic aromatic hydrocarbons form adducts, the nucleotide excision repair enzymes will try to resolve that
  • Xeroderma pigmentosum (XP) proteins assemble at the site of damage - a stretch of nucleotides on either side of the damage are excised
  • People that are defective in these enzymes can’t repair the damage from bulky adducts and are prone to developing cancer
  • Repair polymerases fill the gap and DNA ligase completes the repair
• During or Post-replication repair
  
  • ​Mismatch repair
  • Recombinational repair
  • These proteins check the DNA to make sure that it is ok before the daughter cells bud off in mitosis.

Question 13

Describe the process of excision repair of DNA damage.

Answer 13

• The most electron-rich base is guanine
• NOTE: Adenine is also very electron rich
• If we introduce an electrophile, it will probably target guanine and form a covalent bond - this is a toxic and the cell must remove this
• Base Excision Repair Pathway
  
  • ​DNA glycosylase split/hydrolyses between the sugar and the DNA base
  • Then an AP-endonuclease splits the DNA strand so there is a gap in the backbone
  • DNA polymerase then fills in the missing base (it determines the correct base by looking at the complementary strand)
  • DNA ligase then seals the DNA to form intact DNA
• Nucleotide Excision Repair
  
  • ​Endonuclease makes two cuts in the DNA on either side of the site of damage
  • These patches can be long (100-200 nucleotides) or short (~10-20 nucleotides)
  • Helicase will then remove this patch, leaving the double stranded DNA with a patch missing
  • DNA polymerase then replaces the bases that have been remobed using the complementary strand as a template
  • DNA Ligase then joins the DNA up again
  • This process is energy-demanding and requires a lot of proteins

Question 14

What happens to cells with DNA damage?

Answer 14

• If the DNA damage is excessive, the cells will commit to apoptosis
• However, most problems occur between excessive damage and small amounts of damage
• This could lead to incorrect repair/altered primary sequence
• DNA replication and cell division will then mean that we have fixeddamage in the daughter cells (permanent mutations)
• This can lead to transcriptional and translational problems leading to the formation of aberrant proteins or carcinogenesisis critical targets are mutated (e.g. tumour suppressor genes and oncogenes)

Question 15

How is testing for DNA damage done?

Answer 15

• There is a tiered approach to figuring out whether a chemical agent can cause DNA damage in humans
• You firstly look at the structure of the chemical to see if there are any functional groups that could cause problems
• The simplest way to see whether an agent can cause mutation is to introduce it to bacteria and see whether it causes mutation
• If you can damage the DNA of bacteria then it has potential to damage the DNA of mammals
• Then you test it on Mammalian cells - these have more sophisticated genetic material (e.g. with histones and chromosomes) than bacteria
• Is is then tested in vivo on mammals using bone marrow micronucleus tests and transgenic rodent mutation assays (these are VERY EXPENSIVE)
• Bone marrow is used in such situations because it contains pluripotent stem cells and that gives rise to the cells of the blood
• This means that you can look at the formed elemtns of the blood as a mechanism of what is happening in the bone marrow and what effect the chemical is having on the bone marrow
• It is also a proliferative compartment so you are expanding the potential to cause a mutation.

Question 16

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

Answer 16

• Normally, Salmonella typhimurium is used for this assay
• If we just take the chemical in question and add it to the bacteria, we won’t, generally speaking get a mutation.
• The chemical has to be metabolically activated (which usually requires P450 involvement) - this converts the chemical into something that is damaging
• You incubate the chemical with a preparation of rat liver enzymes (containing P450 enzymes) and that generates the reactive chemical
• The bacteria are genetically engineered so that they can NOT produce histidine (amino acid)
• This means that these bacteria require exogenous histidine to be able to grow
• However, using the chemical mutagens, these cells can be mutated so that they regain the ability to produce histidine, thus meaning that it can grow in the absence of histidine
• So you mix the bacteria with the activated chemical and put them on a plate (which doesn’t have histidine on it)
• Anything that has NOT been mutated will need exogenous histidine to grow and hence will die on the plate
• The bacteria that have been mutated and regained the capacity to produce histidine will grow and survive
• So the more the DNA damaging capability of the chemical, the more colonies will grow in the absence of histidine - this is a very quantitative assay
• This assay also shows a good dose-response relationship

Question 17

Describe the process of detecting DNA damage in mammalian cells (chromosomal aberrations)

Answer 17

• We can look at what is happening to the chromosomes themselves
• Chemicals can cause double strand breaks that leads to fragmentation of chromosomes
• This is very labour intensive investigation

Question 18

Explain what in vitro micronucleus assays show.

Answer 18

• Mammalian cells are treated with the chemical in vitro and allowed to divide
• We are trying to measure the ability of the chemical to break up DNA into fragments, then we can count the fragments
• We need the cell to go through one replication cycle and then stop it when a binucleus is formed
• Cytochalasin-B is used to block cytokinesis and hold the cell in the binucleate stage
• Then the binucleate cells are assessed for the presence of micronuclei
• The kinetochores of the chromosomes can be stained to determine if chemical treatment cause:
  
  • Clastogenicity - chromosomal breakage
  • Aneuploidy - chromosomal loss/change in the number of chromosomes
• Both clastogenicity and aneuploidy can contribute to cancer
• The cell on the left had NOT been damaged by the chemical
• The cell on the right has extra nuclear material - micronucleus - which is not part of the binucleus and has been generated by the chemical.

Question 19

Explain the use of bone marrow micronucleus assays in mice or rats.

Answer 19

• You are using the pluripotent nature of the bone marrow in producing blood cells
• The animals are treated with the chemical and the bone marrow cells or peripheral erythrocytes are examined for the presence of micronuclei
• The erythrocytes normally remove the nucleus during development but it CAN NOT remove small fragments of DNA (e.g. a micronucleus)
• So if the chemical can generate small fragments of DNA as the erythrocytes are formed from the pluripotent stem cells, these fragments will persist.
• The presence of micronuclei in the erythrocytes indicated DNA damage