# Radiation Oncology/Radiobiology/DNA Damage

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Radiation-Induced DNA Damage

## DNA As Radiation Target

* Multiple lines of evidence point to DNA damage being critical component in cell death
** Micro-irradiation studies show that to kill a cell requires much higher dose if cytoplasm only is irradiated vs. if nucleus is irradiated
** Isotopes with short-range emission (e.g I-125) produce efficient killing when incorporated directly into DNA
** Incidence of single strand DNA breaks doesn't result in cell kill, while incidence of double strand breaks and chromosome abnormalities is associated closely with cell kill
** Thymidine analogues (IUdR, BrUdR) modify radiosensitivity when incorporated into chromatin
* However, there is some evidence that radiation damage to cell membranes may also be important, possibly triggering apoptosis
* Finally, there is also evidence for 'bystander effect', where for every micro-targeted cell damaged by RT, neighboring 80-100 would also die and/or show evidence of damage

## DNA Structure

* Base: ring of nitrogen, oxygen, and carbon. Adenine, guanine, thymine, cytosine. Bound to opposite DNA strand via hydrogen bonds
* Nucleoside: Base + deoxyribose sugar
* Nucleotide: Base + deoxyribose sugar + phosphate group(s)
* Backbone: alternating sugar (deoxyribose) and phosphate groups
* Single strand: sequence of nucleotides
* Double strand: two complementary strands bound together via hydrogen bonds
* Chromosome: The entire length of an individual dsDNA; multiple chromosomes are present in a cell. Consists of a centromere and two arms, although under normal circumstances there are not visible as such
* Chromatin: complex of dsDNA and proteins, semi-tightly packed, present in nucleus during interphase. Each chromosome is present as chromatin for packing and ease of access for DNA transcription and repair
* Chromatid: one of two identical copies of DNA making up a chromosome after synthesis during cell division. Tightly packed. Term used as long as chromatids are in contact through centromere. Thereafter they are called daughter-chromosomes

## DNA Damage Overview

* Radiation may damage any of the components of DNA:
** Base damage
** Single strand break (SSB)
** Double strand break (DSB)
** DNA strand cross-links
** DNA-protein cross-links
* Ultraviolet radiation (UV-A) typically causes base damage, such as 8-oxo-G and pyrimidine dimers (e.g. cyclobutane pyrimidine dimer, and 6-4 photoproduct), although can also lead to SSBs. In fact, pyrimidine dimers are usually not found after irradiation with x-rays
* For therapeutic radiation, damage to DNA is caused by energy deposition along the track of the charged particle (electron for x-rays, proton, alpha particle for neutrons)
** Energy is not deposited uniformly along the track, and deposition pattern can be grouped as follows:
** "Spur": diameter of ~4 nm (2x DNA diameter), 100 eV energy deposited, ~3 ion pairs
*** X-rays deposit 95% of their energy via spurs
** "Blob": diameter of ~7 nm, 100-500 eV energy deposited, ~12 ion pairs
*** Neutrons and alpha particles deposit much of their energy via blobs
*** Due to higher number of ion pairs generated, DNA damage can be much more severe than in spurs
* Damage from spur and blob energy depositions can lead to multiple close sites of DNA damage, termed locally multiply damaged site
* Frequency of DNA damage from photon radiation
** A dose that induces on average one lethal event per cell is the D$_0$ dose; statistically ~37% of cells in a population exposed to D$_0$ dose will survive, while other cells will accumulate one or multiple lethal events
** For most mammalian cells, photon D$_0$ is 1-2 Gy
** Number of DNA lesions per cell, immediately after D$_0$ dose:
Events per D$_0$ (1-2 Gy) dose :
| Ionizations | ~100,000/cell |
| Base damage | >1000/cell |
| Single strand breaks | ~1000/cell |
| Double strand breaks | ~40/cell |
| Cell deaths | ~0.63/cell (SF = 0.37) |
* Amount of DNA damage is influenced by the ability of cells to scavenge free radicals produced during ionizations prior to them causing damage, the number of ionizations that are close enough to DNA to damage it, and the effectiveness of DNA repair. As a result, frequency of radiation damage of free DNA is much higher than cellular DNA
* Base damage and single strand breaks
** Typically efficiently repaired
** Do not correlate with cell killing (for example, hydrogen peroxide produces frequent SSBs but doesn't not lead to significant cell kill)
** However, if incorrectly repaired, may lead to alteration in DNA base sequence and thus to mutations
** Frequency of mutations usually increases in dose-dependent manner, but at higher doses, lethal events begin to predominate and frequency of surviving mutations decreases
* Double strand breaks
** Implicated in being primarily responsible for cell killing
** Simple DSB: defined as the occurrence of two single-strand breaks (SSBs) within approximately 6-10 base pairs caused by a single radiation track (PMID 9652804)
** Complex DSB: In addition characterized by a presence of other DNA damage sites (oxidative base lesions, abasic (AP) sites, single strand breaks) in close proximity to the break termini
** Radiation can produce a range of structural complexity DSBs; higher LET radiation produces more complex DSBs due to bleb energy deposition. Low LET radiation is also able to produce complex DSBs
** Strand ends typically have an overhang, that is one strand is several bases longer than the other strand, resulting from an asymmetric break
* Complex double strand breaks
** Occur in ~30% of low LET induced DSBs
** Survival curve shoulder for low LET radiation is thought to reflect the cell's DSB repair capacity
** No survival curve shoulder for high LET radiation may result from the high structural complexity and low repairability of complex DSBs
** It is postulated that complex DSBs may similarly be responsible for majority of cell kill resulting from low LET radiation, after simple DSBs are repaired
** A high degree of associated base damage in complex DSBs may be a factor in poor repair via the non-homologous end-joining pathway (NHEJ)
* Once double strand break has been produced, the broken fragments may have several different fates:
** Rejoin in the original configuration - this leaves no lasting impact at the next mitosis
** Fail to rejoin and remain free - this results in a deletion mutation at the next mitosis
** Rejoin different broken chromosome or fragment - this results in a chromosome/chromatid aberration at the next mitosis, depending on when along the cell cycle the DSB occurred
* Historically, chromosome aberrations are scored at the first metaphase after exposure to radiation. Therefore, depending on when in cell cycle the damage happens, there are two types of aberrations
** Chromosome aberrations - both sister chromatid strands are damaged. Damage happens before chromosome is duplicated; S phase replicates the break in the new sister chromatid
** Chromatid aberrations - single chromatid strand is damaged. Damage happens after chromosome is duplicated; the complementary sister chromatid is undamaged
* There are multiple types of aberrations, some lethal and others not. Please see more at w:Chromosome_abnormalities
** Dicentric (chromosome, lethal): two different chromosomes suffer DSB. The long pieces (containing centromeres) inappropriately join, the two short fragments (acentric) remain free. The long piece containing two centromeres is duplicated. Image
** Ring (chromosome, lethal): one chromosome suffers two separate DSBs, one in each arm. The sticky ends may rejoin to form a ring, while the two short fragments (acentric) remain free. The ring containing a single centromere is duplicated. Image
** Anaphase bridge (chromatid, lethal): Only evident at anaphase, happens after chromosome is duplicated. Both chromatids suffer DSB, and sticky ends join inappropriately. At anaphase, normal separation of chromosomes to opposite poles is not possible
** Symmetric translocation (chromosome, non-lethal): Two different chromosomes suffer DSB. The broken fragments are exchanged, and the sticky ends rejoin. This may result in an oncogene fusion protein (e.g. bcr-abl)
** Deletion (chromosome/chromatid, non-lethal): one chromosome suffers two separate DSBs, on the same arm. The middle fragments falls out, and the outer fragment is inappropriately reattached. The middle fragment is lost at the next mitosis
* Each chromosome is restricted to a specific nuclear domain; as a result, the interactions between breaks are not completely random. Active chromosomes tend to be those with largest surface area (e.g. chromosome 8)
* These radiation-induced DNA events form the basis of the Linear-Quadratic Model

## Double Strand Break Literature

* NIH
** Evaluation of structural organization of radiation-induced DNA double-strand break, produced by 125I-labeled oligonucleotide targeting a particular DNA sequence
** 2005 PMID 15962759 -- "Characterization of a complex 125I-induced DNA double-strand break: implications for repair." (Datta K, Int J Radiat Biol. 2005 Jan;81(1):13-21.)
*** Conclusion: Complex DSBs have high degree of base damage clustering proximal to DSB end
** 2006 PMID 17067210 -- "Molecular analysis of base damage clustering associated with a site-specific radiation-induced DNA double-strand break." (Datta K, Radiat Res. 2006 Nov;166(5):767-81.)
*** Conclusion: Base damage clustering within 8 bases of the I-125 target base. Base lesions were 8-hydroxyguanine, 8-hydroxyadenine, and 5-hydroxycytosine. Also evidence for base damage >24 bp upstream, possibly via charge migration mechanism
* MRC; 2001 PMID 11604075 -- "Computational approach for determining the spectrum of DNA damage induced by ionizing radiation." (Nikjoo H, Radiat Res. 2001 Nov;156(5 Pt 2):577-83.)
** Mone Carlo track simulation. Conclusions:
** (1) Yield of strand breaks per unit absorbed dose is nearly constant over a wide range of LET
** (2) Majority of DNA damage is of simple type, though majority of SSBs are accompanied by at least one base damage
** (3) For low energy electrons, ~20-30% of DSBs are complex DSBs. This increases with LET, reaching ~70% for alpha particles
** (4) Extent of damage in local hit region of the DNA is limited to few base pairs
* Columbia; 1992 PMID 1351522 -- "Constraints on energy deposition and target size of multiply damaged sites associated with DNA double-strand breaks." (Brenner DJ, Int J Radiat Biol. 1992 Jun;61(6):737-48.)
** Locally multiply damaged sites (LMDS) are energy depositions of 2-5 ionizations localized in 1-4 nm diameter
* UCSD; 1990 PMID 1971840 -- "The yield of DNA double-strand breaks produced intracellularly by ionizing radiation: a review." (Ward JF, Int J Radiat Biol. 1990 Jun;57(6):1141-50.)
** DNA dsb linearly related to dose; DNA damage per dose constant among cell types
** Variations in radiosensitivity assigned to difference in repair speed and/or accuracy

## Detection of DNA Damage
### Base damage

* High pressure liquid chromatography (HPLC)
* Tritium release from labeled thymine
* Phosphate release
* Fluorescent labeling

### Strand breaks

* For most of the assays, DSBs are evaluated in neutral pH (7-10) solutions and SSBs are evaluated in alkaline pH (~12) solution
* Damage detected by these assays is typically repaired within 24 hours
* Sucrose gradient segmentation - centrifuge
** Sucrose solution with gradient varying from 5%-30%
** Cell DNA radiolabeled (e.g. $^{14}$C)
** Cell lysis induced, DNA released (depending on solution as single strand or double strand) to sucrose solution
** Sucrose solution with DNA then centrifuged
** Larger DNA fragments travel differently through the sucrose solution than small fragments
** Fractions of the fluid are then collected and assayed for $^{14}$C to evaluate amount of DNA present at each gradient level
** Strand break frequency is estimated from distribution of fragment size
* Nucleoid sedimentation - centrifuge
** Sucrose gradient solution is used
** Cells are lysed at neutral pH in the presence of high salt concentration and non-ionic detergent
** Interphase nucleus opens up, and chromatin strands are revealed. The individual broken strands are called nucleoids, and consist of supercoiled DNA still wrapped around residual protein structures
** Single strand DNA breaks allow the chromatin fragments to relax the supercoil and enlarge
** Differently sized chromatin fragments travel differently through the sucrose solution
** Sometimes, a fluorescent dye is added, and resulting halos can be measured by microscopy (= the halo method)
* Neutral filter elution - elution
** Filter with pore size ~2 mm (DNA fiber has diameter ~25nm, 100x smaller)
** Cell DNA is radiolabeled (e.g. $^{14}$C)
** Cell lysis induced, DNA released (depending on solution as single strand or double strand) on top of filter
** Elution buffer solution is used to wash DNA fragments through the filter
** Rate of DNA elution is related to size of DNA fragments
** Double strand breaks are assayed at pH 7.4-9.6, single strand breaks are assayed at more alkaline pH 12.3
* Pulsed-field gel electrophoresis (PFGE) - electrophoresis
** Agarose gel
** Cell DNA is radiolabeled (e.g. $^{14}$C) or DNA-specific fluorescent dye is used after the electrophoresis
** Cell lysis induced, DNA released (depending on solution as single strand or double strand) to agarose gel
** Fragments of DNA carry a net negative charge
** Electric field is applied to the agarose gel, and DNA fragments travel at speed inversely related to their size
** Constant field electrophoresis can work, but separation of fragments is improved by pulsing the field at different directions to axis of migration
* Single-cell gel electrophoresis (Comet assay) - electrophoresis
** Cells are embedded in agarose gel and then lysed to release DNA in-situ
** Electric field is applied, and DNA fragments migrate away from the DNA mass in the nucleus outward
** The shape of the resulting migration structure resembles a comet, hence the name
** Double strand breaks are assayed at neutral pH, while single strand breaks are assayed at alkaline pH
** High sensitivity to SSB, though not as much for DSB
* Gamma-H2AX assay - immunohistochemistry/flow cytometry
** IHC is very labor intensive and not easily implemented in a routine practice, but allows detection of ~10X lower level of DNA damage
** This assay is a highly sensitive method for detecting presence (and/or repair) of individual DSBs

### Chromosome aberrations

* Stable chromosome aberrations can be present for months - years after exposure
* Visual observation - at the first metaphase after exposure to ionizing radiation
* Chromosome aberration assay - immunohistochemistry via FISH painting

## Chromosome Aberrations in Human Lymphocytes

* Used widely as biomarkers of radiation exposure
* Incidence of dicentrics and rings (see above) in peripheral blood lymphocytes is scored after total body irradiation exposure
** Dose-response fits linear quadratic relationship
** Doses as low as 0.25 Gy can be detected, which can be used for evaluation of "black film badges" or potential accidents
* Damage kinetics
** Terminal deletions - single hit - linear response
** Dicentrics, translocations, rings, interstrand deletions - two hits - quadratic response
* Radiation exposure can be tracked over time
** Mature lymphocytes have life span of ~1500 days (~4 years) and are slowly eliminated from the peripheral blood. Therefore, yield of asymmetric aberrations (dicentrics, rings) in mature lymphocytes declines over time
** Stem cell/precursors lymphocytes also experience DNA damage, which has different time course depending on type
** Unstable aberrations: Asymmetric aberrations (e.g. dicentrics, rings) kill the cell, and are not passed on to progeny. Their yield decreases over time
** Stable aberrations: Symmetric translocations may persist indefinitely, as they are propagated through the progeny. Their yield remains stable over time
** Frequency of translocations after >50 years in Hiroshima survivors correlates well with total body dose received at that time