DNA Damage

Question 1

Give 6 types of DNA damage - list them

Answer 1

1. Base dimers and cross-linking
2. Base hydroxylations
3. Abasic sites
4. Single strand breaks
5. Double strand breaks
6. DNA adducts and alkylation

Question 2

What happens when you get base dimerisation and cross-linking?

Answer 2

• DNA molecules are being chemically linked up
• Form of DNA damage

Question 3

What happens when you get base hydroxylation (a type of DNA damage)?

Answer 3

• An oxidative reaction occuring on one of the DNA bases and this necessitates DNA repair
• During this DNA repair, the DNA could get mutated

Question 4

What happens in abasic sites (a type of DNA damage)?

Answer 4

• During DNA repair, sometimes one of the DNA bases is excised while the sugar phosphate backbone is maintained
• During replication, the missing base will be a problem

Question 5

What happens in single strand breaks (a type of DNA damage)?

Answer 5

• Very common and useful
• There are many physiological enzymes that cause single strand breaks for example topoisomerase causes the relaxing and unwinding of the DNA so that the DNA can be accessed and then also the strand can be re-annealed
• Single strand breaks are not a big deal

Question 6

What happens in double strand breaks (a type of DNA damage)?

Answer 6

• These are disastrous
• When the double strand breaks, the DNA can drift off which is intolerable
• There are DNA repair mechanisms to prevent XS damage in case of double strand breaks, but these often introduce mutations

Question 7

What happens in DNA adducts and alkylation (a type of DNA damage)?

Answer 7

• Some chemicals can be metabolically activated into electrophiles
• So they become attracted to the electron-rich DNA (lots of electrons in the nitrogen in the bases)
• The electrophiles bind to the DNA and form a covalent bond
• The binding of another molecule to the DNA molecule is harmful because during replication, DNA polymerase will not be able to recognise the base due to the chemical adduct

Question 8

What are the basic principles behind what happens in phase 1 and 2 reactions, give examples of these reactions (3 examples for phase 1 reactions and 6 examples for phase 2 reactions), and what usually carries out phase 1 reactions - or a particular type of phase 1 reactions?

Answer 8

PHASE 1:

• Addition of a functional group (introduction or unmasking of a functional group)

1. Oxidation
2. Reduction
3. Hydrolysis

• Mainly cytochrome P45O mediated (oxidation) - it has a broad substrate specificity and oxidises mainy chemicals

PHASE 2:

• Conjugation of a polar group onto the phase 1 functional groups which have been added or unmasked
• This is in order to make the lipophilic molecule more polar and therefore water soluble so it can be more readily excreted

1. Methylation
2. Acetylation
3. Sulphation
4. Glucoronidation
5. Amino acid conjugation
6. Glutathione conjugation

Question 9

Where can polycyclic aromatic hydrocarbons be found?

Answer 9

IN SMOKE

• In common environmental pollutants
• From combustion of fossil fuels
• From combustion of tobacco (smoking)

Question 10

What type of molecule is benzopyrene and describe the stepwise process in how it can cause DNA damage and is therefore carcinogenic?

Answer 10

• Benzopyrene is a polycyclic aromatic hydrocarbon
• `It is oxidised by CYP450 to produce an epoxide / oxide (benzo(a)pyrene-7,8-oxide)
• This metabolite is unreactive and unstable and potentially damaging, it is an electrophile so can cause DNA damage
• But this is metabolised further by epoxide hydrolase into a non-toxic dihydrodiol
• BUT
• This non-toxic dihydrodiol metabolite is further metabolised by CYP450 again into a very reactive diol epoxide
• This diol epoxide becomes +vely charged and seeks out electrons
• Thus it is attracted to the electron rich DNA
• Therefore this results in DNA adducts
• The DNA adducts starts the mutation process

Question 11

Aflatoxin B1…

1) Where is it found / how is it formed?
2) What type of cancers does it cause? Why specifically here?
3) Outline the process of how it is carcinogenic

Answer 11

1)

• Formed by Aspergillus Flavus mould found on poorly stored grain such as rice and peanuts

2)

• Liver carcinogen - hepatocellular carcinoma
• Targets the liver because it is activated by CYP450 found only in the liver

3)

• Aflatoxin B1 is metabolised by CYP450 in the liver into aflatoxin B1-2,3-epoxide
• This binds the N7 position on a guanine to form DNA adducts
• So DNA repair machinery comes along to repair the DNA
• When its fixed, its fixed inappropriately, leading to DNA damage

Question 12

Why can polycyclic aromatic hydrocarbons cause cause cancer anywhere, whereas aflatoxin B1 has a very localised area where it is carcinogenic (and where is this area)?

Answer 12

• Because CYP450 which is involved in some steps of the metabolism of polycyclic aromatic hydrocarbons can be found throughout the body, so the final toxic metabolite can be formed
• Whereas the CYP450 which metabolises aflatoxin B1 into aflatoxinB1-2,3-epoxide is only found in the liver - so it is linked to hepatocellular carcinoma

Question 13

2-Napthylamine

1) Where is this chemical commonly found?
2) What type of cancers is it carcinogenic for?
3) Outline the process of how it is carcinogenic

Answer 13

1)

• In dyes

2)

• Bladder cancer

3)

• CYP450 metabolises 2-Napthylamine to convert the amino acid to form a hydroxylamine
• Hydroxylamines are reactive
• These hydroxylamines are glucuronidated by glucuronyl transferase
• In the acidic conditions of the urine, the molecule rearranges to form a nitrenium ion which is an electrophile and therefore forms a DNA adduct

Question 14

How is solar (UV) radiation carcinogenic?

Answer 14

• UV light induces pyrimidine dimers. Remember pyrimidines include cytosine, uracil and thymine
• If there are 2 pyrimidines nearby and are in the presence of UV radiation, they can covalently link
• The cell tries to repair this, but in doing so, a mutation is introduced
• SKIN cancer

Question 15

Give 3 examples of ionising radiation and how is ionising radiation carcinogenic?

Answer 15

1. Gamma radiation
2. X-rays
3. Beta-particles

• They generate free radicals including oxygen free radicals such as superoxide (O^{<strong>.</strong>}) and hydroxyl (HO^{<strong>.</strong>}) free radicals
• So this unpaired electron on the free radicals make them very reactive, they seek out electrons to pair with so they are attracted to the electron rich DNA
• This causes DNA damage in many ways, including causing both single and double stand DNA breaks and generating abasic, apyrimidine DNA sites or even introducing base modifications

Question 16

1) What are free radicals and why are they damaging to DNA - don’t need to detail the exact mechanisms
2) Give 2 types of free radicals
3) Give one way that these free radicals can be generated

Answer 16

1)

• Molecules or atoms with an unpaired electron which makes it very electrophilic and reactive so interact with the DNA

2)

• Superoxide (O^{<strong>.</strong>})
• Hydroxyl free radical (HO^{<strong>.</strong>})

3)

• Ionising radiation can generate free radicals

Question 17

Outline the process of how oxygen free radical attack can damage DNA

Answer 17

• They can cause DNA damage by 6 mechanisms:
  1. Double (damaging) DNA strand breaks
  2. Single DNA strand breaks (not very damaging)
  3. Generate abasic (apyrimidic) sites

Introducing base modifications including:

4. Ring-opened guanine and adenine
5. Thymine and cytosine glycols
6. 8-hydroxyadenine + 8-hydroxyguanine (mutagenic)

Question 18

What is the role of p53 in cellular stress?

Answer 18

• p53 is a crucial tumour suppressor gene (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 activates proteins that help repair the problem
• If there is SEVERE stress, p53 can activate an apoptotic pathway by interacting with apoptosis proteins

Question 19

List the 4 different types of DNA repair mechanisms - you do not need to detail them at this point

Answer 19

1. Direct reversal of DNA damage
2. Base excision repair
3. Nucleotide repair
4. During or post-replication repair

Question 20

Detail 2 examples of direct reversal of the DNA damage

Answer 20

1. Photolyase splits cyclobutane pyrimidine-dimers formed as a result of UV exposure which initially causes the base dimerisation, in order to recover the pyrimidines
2. Methyltransferases and alkyltransferases remove alkyl groups from DNA bases to repair the alkylation of DNA - a form of DNA damage

Question 21

Detail what type of DNA damage base excision repair is particularly effective in repairing and the process of what happens in base excision repair

Answer 21

• Particularly useful in repairing abasic (apurinic / apyrimidic) damage

1. DNA glycosylase splits / hydrolyses between the base and the sugar phosphate backbone to release the base
2. Then an AP endonuclease splits the DNA strand so there is a gap in the sugar-phosphate backbone
3. DNA polymerase fills in the missing base (determines the correct base using the complementary strand)
4. DNA ligase then seals the DNA to form intact DNA

Question 22

Detail what type of DNA damage nucleotide repair is particularly effective in repairing and the process of what happens in nucleotide repair

Answer 22

• Particularly useful in treating DNA damage by DNA adducts

1. Endonuclease makes 2 cuts in the DNA at either side of the DNA damage (or adduct)
2. Helicase then removes this patch, leaving the double stranded DNA with a patch missing
3. DNA Polymerase replaces the removed bases using the complementary strand as a template
4. DNA ligase then joinds the DNA strands back together

• This is a highly-energy demanding process and requires protein

Question 23

Describe the 3 fates that can occur upon DNA damage induced by a carcinogen

Answer 23

1. If the damage is too great, the cell is committed to apoptosis
2. If there is very little damage and it is manageable, then the DNA repair mechanism may be able to completely repair the damage without issue
3. Sometimes the DNA repair mechanism can be incorrect and introduce error such as altered primary sequence (mutation). This mutation can be fixed upon DNA replication and cell division. The mutations can lead to transcriptional and translational problems leading to the formation of aberrant proteins or carcinogenesis is critical targets are mutated (e.g. tumour suppressor genes and oncogenes

Question 24

Outline the 5 steps in order for methods to see if a particular agent can cause DNA damage - you don’t need to describe them yet, just mention them

Answer 24

1. Structural analysis - are there any functional groups which you might expect may cause damage?
2. In Vitro BACTERIAL gene mutation test e.g. Ames test with S.Typhimurium
3. In Vitro MAMMALIAN CELL assay e.g. chromosomal aberration or micronucleus tests
4. In Vivo MAMMALIAN assay e.g. bone marrow micronucleus test or transgenic rodent mutation assay
5. Investigative In Vivo MAMMALIAN assays

Question 25

Describe the bacterial Ames test for mutagenicity of chemicals, starting with the name of the bacteria that is commonly used in the test

Answer 25

* Salmonella typhimurium * Add the chemical to be tested, rat liver enzyme preparation and the *S.Typhimurium* bacterium which has been modified so it cannot produce histidine into a histidine free medium on an agar plate * The rat liver enzyme preparation is to provide enzymes such as CYP450 that aren't produced by the bacterium and may metabolise the test substance and convert it into reactive metabolites * Histidine is an amino acid that is essential for the survival of the bacterium, so if you do not provide exogenous histidine through the medium, it must produce histidine endogenously in order to survive * If the substance or its reactive metabolites are indeed mutagenic, they will restore the bacterium's ability to synthesise histidine so will survive and form colonies on the histidine-free medium * The more the DNA damaging capability of the chemical, the more colonies grow in histidine absence * This is a very quantitative assay, and also shows a good dose-response relationship

Question 26

Describe how you can do in vitro mammalian cell assays - describe the test looking at chromosomal abberrations to test for mutagenicity of substances

Answer 26

* You incubate the cells with the DNA-damaging chemical in the presence of liver S9 - an enzyme preparation * Then, you do karyotyping or you look at the chromosomal structure * If chromosomes are damaged in any way, it indicates that the chemical has damaged the mammalian cell. You can look for chromatid exchange, chromatid gaps etc

Question 27

Describe the *In Vitro* mammalian cell assay - micronucleus assay testing for substance mutagenicity

Answer 27

* Mammalian cells are exposed to the substance you are trying to see is mutagenic in vitro * We allow the passage of one cycle (almost) * We stop cell division when a binucleus is formed - Cytochalasin-B is used to arrest it at cytokinesis at the binucleate stage * Then the binucleate cells are assessed for the presence of micronuclei which are bits of chromosomal material that have broken off and away from the nucleus - thus indicate the ability of the chemical to break up DNA The kinetochores can also be stained to determine if chemical treatment caused the following (both of which result in cancer): * Clastogenicity: chromosomal breakage * Aneuploidy: chromosomal loss/change in the number of chromosomes

Question 28

Describe the bone marrow micronucleus assay - a form of in vivo mammalian assay testing for substance mutagenicity

Answer 28

* You are using the pluripotent nature of the bone marrow in producing blood cells * The animals are treated with the chemical * Bone marrow cells or peripheral erythrocytes are examined for the presence of micronuclei * The erythrocytes normally remove the nucleus during development * But they CAN NOT remove small fragments of DNA (e.g. a micronucleus) * 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 indicates DNA damage