DNA Damage Flashcards
What are the two types of DNA damage?
- Spontaneous alterations and damage to DNA
2. Environmental damage to DNA
What is DNA damage most commonly caused by?
Chemicals or radiation
How are people exposed to chemical carcinogens?
It’s a result of being exposed due to occupation, pollution in the ambient environment, lifestyle choices or pharmaceutical use.
What do chemical carcinogens act through?
They act through “genotoxic” or DNA damaging mechanisms, which involve covalent binding of the chemical to DNA (DNA adduct formation)
What is a necessary requirement for tumor induction when exposed to cancer carcinogens?
They’re typically frequent and/or chronic over the years which results in the accumulation of DNA damage or DNA adduct formation
What have studies in animal models indicated about environmental DNA damage?
The ability to reduce DNA damage will also result in the reduction of tumour risk
What was the hypothesis determined by the animal model studies?
Individuals having the highest levels of DNA adducts may have an increased cancer risk, compared to individuals with the lowest levels of DNA adducts
What is a DNA adduct?
A segment DNA that is bound to a cancer causing agent
What did Poirier review in 2012?
He reviewed 12 investigations showing a 2- to 9-fold increased risks for cancer in individuals having the highest DNA adduct levels, compared matched individuals with the lowest DNA adducts
What other preliminary evidence did the studies reviewed by Poirier provide?
It provided preliminary evidence that multiple types of DNA adducts combind, or DNA adducts combined with other risk factors (eg., infection or inflammation), may be associated with more than 10-fold higher cancer risks, compared to those found with a single carcinogen
What did the data from the preliminary evidence reviewed by Poirier suggest?
It suggested that a reduction in human DNA adduct level is likely to produce a reduction in human cancer risk
What reactive oxygen species are produced by normal metabolism?
Superoxide and hydrogen peroxide
What is NOX?
NAD(P)H oxidase (NOX) is a membrane bound enzyme that macrophages use to produce O2- to kill bacteria
Is NOX upregulated or downregulated in TAMs?
Upregulated
How does the Fenton reaction lead to DNA damage?
Reduced transition metals react with hydrogen peroxide to produce hydroxyl radicals which have the capacity to damage macromolecules (eg., DNA)
What are the three ways radical oxides, from the Fenton reaction, damage biomolecules?
- Scission: DNA double-strand breaks
- Modification: 8-oxoguanine, HNE, etc.
- Cross-links: thymine-tyrosine cross-links, etc.
What cancer causing mutations of the genome are due to damage of biomolecules?
- Amplification
- Deletion
- Point mutation
- Translocation (fusion gene)
What does oxidation of DNA lead to?
DNA damage and mispairing
How can Chromosome Aberrations be detected?
- Microscopy detection
2. Importance of double strand breaks
How can micronuclei be detected?
- Microscopy detection
2. Flow cytometry detection
How can single strand breaks be detected?
- Comet assay (single cell gel electrophoresis)
- Alkaline elution
- Gel electrophoresis
- PCR assays
What was the result of antioxidant supplementation (alpha-tocopherol, beta-carotene and vitamin C) in smokers and non-smokers?
- There were decreases in oxidative DNA damage in human blood lymphocytes.
- Sites sensitive to Endonuclease III, an enzyme that excises oxidized pyrimidine bases from DNA, decreased
What is the result of gamma radiation?
DNA double strand breaks
How was the DNA, in the experiment by Dr.Chantal Courtemanche, treated?
- Fractions of DNA was actively release
- cells were irradiated either in agarose (no repair) or in culture (possible recombination repair)
What is significant about hypoxia and its effect on DNA?
- It’s an oxidant stressor
- The microenvironment is supported by HIF1-alpha and angiogenesis
- > induces DNA damage
What does DMTU do?
- An antioxidant
- Attenuates hypoxia-induced DNA damage
What is an example of DNA base damage?
Modified guanines: oxo^8-dG
What are the seven sources of oxidative DNA damage?
- Endogenous metabolism
- Inflammation
- Smoking
- Cancer
- Ionizing radiation
- Exercise
- Xenobiotics
How many oxidation products of DNA bases are possible?
18 adducts are possible
What are the three types of oxidized guanines?
- 8-oxo-2’-deoxyguanosine (oxo^8dG)
- 8-oxoguanosine (oxo^8G)
- 8-oxoguanine (oxo^8Gua)
Why is oxidized guanine a problem?
It is a modification to the DNA that could cause problems in DNA synthesis due to improper base pairing
What is the advantage to Gas Chromotography Mass Spectrometry (GC-MS) detection of modified bases?
Can detect an array of base damage
What is the disadvantage to Gas Chromotography Mass Spectrometry (GC-MS) detection of modified bases?
Must run at a high temperature
What is the advantage of High Performance Liquid Chromatography - Electrochemical (HPLC-EC) detection of modified bases?
Can be done at room temperature minimizing artifactual damage
What is the disadvantage of HPLC-EC detection of modified bases?
Can only detect modified bases that give a clear EC signal
What does Epigallocatechin o-Gallae (EGCG) do?
Inhibits neutrophil infiltration and oxidative DNA damage in phorol ester-treated mouse skin
What important discovery was determined from the EGCG experiments?
Tumour numbers and volume correlate with oxidative DNA damage but if you block the oxidative DNA damage, you can block the amount of tumour
What is EGCG?
A gallate produced in plants
- The reason that eating more fruits and vegetables can be a cancer preventative (increases EGCG)
What are the advantages of Antibody detection?
High throughput
What are the disadvantages of Antibody detection?
Not analytical, mostly qualitative in nature up to this point
What is Ferric nitrilotiracetate-induced oxidative DNA damage in renal proximal tubule cells associated with?
Necrotic cell death
What is the advantage to Post labeling methods for detecting base damage?
It’s sensitive
What is the disadvantage to Post labeling methods for detecting base damage?
Must use a radioactive nucleotide for detection
What happens when Uracil is incorporated into DNA and not repaired?
It causes chromosome breaks
- the same way as that of radiation induced chromosome breaks
- lesions in opposing DNA strands are excised by base excision repair, which causes a chromosome break
How can you detect uracil incorporation into DNA?
GC-MS with negative Cl for the detection of uracil in DNA
What are AP sites?
- Apurinic and apyrimidinic (AP or abasic) sites
- lesions in DNA
- formed either spontaneously or as intermediates during the course of base excision repair of oxidized, deaminated or alkalated bases
Where are oxidative DNA lesions with mutagenic potential formed?
In inflammation-related cancer cells
What was the mechanism proposed by Ohnishi et al?
The generation of cancer stem cells by inflammation:
- Chronic inflammation results in the production of ROS and RNS
- ROS and RNS cause both DNA damage and tissue injury in the stem cells
- DNA damage results in genomic instability and mutation
- The mutation and damaged stem cells result in cancer stem cells that lead to carcinogenesis
What does chronic inflammation by infectious agents, inflammatory diseases, and other factors cause and how does it cause this damage?
ROS/RNS generation results in various types of damage to nucleic acids, proteins and tissues
What can tissue injury during chronic inflammation activate?
Progenitor/stem cells for regeneration
In the progenitor/stem cells made for regeneration, what can ROS/RNS generate?
Due to the ROS/RNS causing multiple mutations, they may generate mutant stem cells and cancer stem cells which leads to carcinogenesis.
Where was 8-nitroguanine formed?
It was formed in stem cell marked positive cells in infection-associated cancer tissues
