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
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
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 D0 dose; statistically ~37% of cells in a population exposed to D0 dose will survive, while other cells will accumulate one or multiple lethal events
For most mammalian cells, photon D0 is 1-2 Gy
Number of DNA lesions per cell, immediately after D0 dose:
Events per D0 (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)
Evaluation of structural organization of radiation-induced DNA double-strand break, produced by 125I-labeled oligonucleotide targeting a particular DNA sequence
2005PMID 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
2006PMID 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; 2001PMID 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; 1992PMID 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; 1990PMID 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
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. 14C)
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 14C 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. 14C)
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. 14C) 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
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