Cancer 5 Flashcards
1
Q
what is the nature of the DNA bases?
A
- they are flat carbon rings
- they are chemically reactive species able to react with other molecules
- they contain double bonds
- they are easily mutated
2
Q
what is deamination?
A
- The primary amino groups of nucleic acid bases are somewhat unstable
- deamination is the removal of an amine group
- They can be converted to keto-groups
- Other deamination reactions include conversion of adenine to hypoxanthine, guanine to xanthine, and 5-methyl cytosine to thymine.
3
Q
what causes hyper-reactive oxygen to be formed?
A
bases can undergo reactions which produce hyper-reactive oxygen as by-products
eg. singlet oxygen, peroxide radicals, hydrogen peroxide, and hydroxyl radicals
4
Q
what causes modifications of DNA to be formed?
A
- ionising radiation
- hyper- reactive oxygen species
5
Q
what is a common oxidation reaction?
A
- A common product of thymine oxidation is thymine glycol
6
Q
what happens to the glycol?
A
- they react with chemical species which favor it
- these reactants can be carcinogens
- the addition of a larger molecule produces what we call an ‘adduct’
7
Q
What is photodamage to the DNA?
A
- Ultraviolet light is absorbed by the nucleic acid bases
- the resulting influx of energy can induce chemical changes
- photoproducts are formed as a consequence of bond formation between adjacent pyrimidines within one strand.
8
Q
what are the types of DNA damage?
A
- base pair mismatch
this is when one base is normal and the matching base is abnormal it causes a slight bulge
- thymine dimer
- radioactive damage
9
Q
what can cause damage to DNA?
A
- endogenous means from within the body
eg mitochondria produce reactive oxygen species that have the ability to damage DNA
- Diet is strongly associated with cancer (about 40=45% of human cancers)
- Medical treatments, such as radiotherapy, can also damage DNA and increase the risk of cancer
10
Q
why is DNA damage important?
A
- DNA damage can lead to mutation
- mutation can lead to cancer
- •Damaging DNA is an important strategy in cancer therapy
11
Q
how can carcinogens cause DNA damage?
A
- Base Dimers and Chemical Cross=Links
- Base Hydroxylations
- Abasic Sites
- Single Strand Breaks
- Double Strand Breaks
- DNA Adducts and Alkylation
12
Q
explain base dimers and chemical cross-links ?
A
- This is where the DNA molecules are being chemically linked up
13
Q
explain base hydroxylations?
A
- An oxidative reaction occurring on one of the DNA bases and this can cause problems
- This could mean that the DNA has to get repaired and during the repair process, it could become mutated
14
Q
explain 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, the missing base will cause problems
15
Q
explain single-strand breaks?
A
- 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
16
Q
explain double-strand breaks?
A
- 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
17
Q
explain DNA adducts and alkylation?
A
- This is generally the type of damage that is caused by chemicals
- Some chemicals tend to be metabolically activated into electrophiles
- 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
- this causes issues in DNA replication due to the binding of the big bulky chemical
- In short, DNA polymerase cannot recognise the base because of the chemical adduct
18
Q
what are polycyclic aromatic hydrocarbons?
A
- Polycyclic aromatic hydrocarbons are common environmental pollutants
- they are present in smoke
19
Q
overview drug metabolism in the body?
A
- 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
overall purpose is to make something that is lipophilic more polar to get rid of it
20
Q
what are the main enzymes in phase 1?
A
- Cyrochrome P450 enzymes
- responsible for oxidising chemicals
21
Q
what is the two-step Oxidation of Benzo[a]pyrene (B[a]P)?
A
22
Q
explain why the two-step oxidation of Benzo[a]pyrene (B[a]P) is dangerous?
A
- B[a]P is a substrate for CYP450, which oxidizes it to form an oxide (Benzo[a]pyrene-7,8-oxide) (toxic)
- 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 (this is NOT toxic)
- the non-toxic dihydrodiol metabolite is also a substrate for P450
- So P450 converts this non-toxic metabolite into another oxide
- This is very reactive and is desperate to find some electrons to react with
- the best source of electrons is DNA, so DNA adducts are formed
23
Q
explain why the metabolism of aflatoxin B1 is dangerous ?
A
- aflatoxins are produced from fungi aspergillus fungi
- grow on foodstuffs that are not maintained well and peanuts
- aflatoxin B1 is not a carcinogen
- cytochrome P450 converts it to an apoxite
- this apoxite is very reactive with DNA
- it reacts with the guanine in DNA at the N7 position
- we end up with an adduct formation on the guanine that will cause mutation
- this is very carcinogenic for hepatic cancers
24
Q
explain why the metabolism of 2-naphthylamine is dangerous?
A
- 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
- 2- naphthylamine is a substrate for CYP450, which converts the amino group to form a hydroxylamine
- Hydroxylamines are reactive
- in the liver when this reactive hydroxylamine is formed it is glucuronidated ( detoxifying reaction)
- This glucuronidation 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 acidic conditions rearranges to form a positively charged nitrogen
- 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
25
how does solar radiation cause cancer?
* UV radiation can lead to the formation of pyrimidine dimers
* if there are two pyrimidines next to each other in the presence of UV radiation they can covalently link
* this might form skin cancer
26
how does ionizing radiation cause cancer?
* Examples of ionising radiation: gamma, X=ray, beta particles
* They can all generate free radicals
* Oxygen- free radicals are produced by normal biochemistry and there are good defence mechanisms for dealing with them = however, ionising radiation can overwhelm the defence mechanisms
27
what is a superoxide radical?
* a superoxide radical is very powerful
* this is a molecule of oxygen that has an extra electron so it is very reactive
28
what is a hydroxyl radical?
* a hydroxyl group that has grabbed an extra electron
* this is even more reactive than the superoxide radical
* these radicals are electrophiles
* DNA is electron-rich
29
how do oxygen free radicals attack DNA?
* single strand breaks are not very damaging
* double-strand breaks are more dangerous
* when the base has been oxidized by an oxygen free radical and the DNA repair enzymes come and cut out the base itself it leaves behind the sugar- phosphate backbone with gaps
* these gaps are called apurinic / apyrimidic sites
* You can also get base modifications:
Ring- opened guanine + adenine
Thymine + cytosine glycols
8- hydroxyadenine + 8- hydroxyguanine (mutagenic)
30
what is the role of P53 in dealing with cellular stress?
* P53 is a crucial tumour suppressor gene
* it is normally tied to MDM2 which keeps the P53 inactive
* when it is released from MDM2 it forms a dimer which activates many different pathways
* If we have mild physiological stress = p53 carries out 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 directly interacting with apoptosis proteins
31
what are the 4 main types of DNA repair?
* direct reversal of DN damage
* base excision repair
* nucleotide excision repair
* during or post replication repair
32
explain direct reversal of DNA damage?
**Photolyases**
* specific group of enzymes called **photolyases** can reverse cytlobutane- pyrimidine dimers
* Photolyase looks specifically for cytlobutane=pyrimidine dimers and cuts them to restore the normal sequence
* this allows repair of the DNA to occur
**Methyltransferases and alkyltransferases**
* Methyltransferases and alkyltransferase remove alkyl and methyl groups from bases to restore the identity of the base
* these enzymes then destroy themselves
33
what is DNA mismatch repair?
* mismatch repair involves scrutinisation of of DNA for apposed bases that do not pair properly
* Mismatches that arise during replication are corrected by comparing the old and new strands (proofreading)
* this is in place as there is a large rate of error that takes place in DNA replication
34
why does guanine often form adducts?
* The most electron=rich base is guanine
* if we introduce an electrophile it will probably target guanine and form a covalent bond, this is toxic and the cell must remove this
35
what is the base excision repair pathway?
* DNA glycosylase 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
* DNA Ligase then seals the DNA to form intact DNA
36
what is 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 removed 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
37
what is Xeroderma Pigmentosum?
this is the main disease to remember
* this is a disease in which nucleotide excision repair does not work properly
* causes severe light sensitivity
* severe pigmentation irregularities
* early onset of skin cancer and GI cancer at high incidence
* frequent neurological defects
* this disease is not curable - only managed, must AVOID UV night
38
what is Trichothiodystrophy?
this is a disease in which nucleotide excision repair does not work properly
* sulphur deficient brittle hair
* facial abnormalities
* short stature
* ichthyosis (fish-like scales on the skin)
* light sensitivity in some cases
39
what is cockaynes syndrome?
this is a disease in which nucleotide excision repair does not work properly
## Footnote
* dwarfism
* light sensitivity in some cases
* facial and limb abnormalities
* neurological abnormalities
* early death due to neurodegeneration
40
when do DN A double-strand break repairs happen?
* Under physiological conditions during somatic recombination and transposition
* During Homologous Recombination.
* As a result of ionizing radiation and oxidative stress induced DNA damage.
41
how to join DNA after DN double-strand break repair?
there are two main methods
homologous recombination and nonhomologous DNA end-joining
**homologous recombination =**
* Direct joining of the broken ends.
* This requires proteins that recognize and bind to the exposed ends and bring them together for ligating.
* this happens with complementary nucleotides
**non** **homologous recombination = (more complicated)**
* 3 DNA ends are picked up by KU proteins
* KU holds the DNA together and you force the DNA to link up
42
what are the estimated rates of endogenous damage and repair?
* It appears that human cells have plenty of spare capacity to deal with both endogenous and exogenous damage
* errors occur, especially with greater age
* If the damage is poorly repaired, then there is greater risk of carcinogenesis
43
what happens if the cell has too much damage?
If the DNA damage is excessive, the cells will commit to apoptosis
44
at what point do most issues occur resulting in cancer?
* However, most problems occur between excessive damage and small amounts of damage
* This could lead to incorrect repair
* this results in fixed damage in the daughter cells (permanent mutations)
45
how to test for DNA damage in cells?
* 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 it damages bacteria DNA it might damage human DNA
* Then you test it on Mammalian Cells
* then you test it on mammals using bone marrow micronucleus tests
* bone marrow is used because it contains pleuripotent stem cells
* This means that you can look at the formed elements of the blood as a mechanism of what is happening in the bone marrow
46
what is the bacterial Ames test for mutagenicity of chemicals?
* Normally, Salmonella typhimurium is used for this assay
* we take chemical and make it metabolically active
* to do this we 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 any histidine on it)
* Anything that has NOT been mutated will need exogenous histidine to grow and hence will die on the plate
* **So the more the DNA damaging capability of the chemical, the more colonies will grow in the absence of histidine**
47
how to detect DNA damage in mammalian cells?
* 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
48
how to use in vitro micronucleus Assays?
* 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
* Then the binucleate cells are assessed for the presence of micronuclei
* The kinetochores of the chromosomes can be stained to determine if chemical
treatment caused:
o Clastogenicity = chromosomal breakage
o Aneuploidy = chromosomal loss/change in the number of chromosomes
* both of these cause cancer
* The cell on the left has NOT been damaged by the chemical
* The cell on the right has extra nuclear material
49
how to carry put bone marrow micronucleus in mice or rats?
* 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 (micronucleus)
* so presence of micronucleus indicates DNA damage which could cause cancer
