Radiobio part 2 Flashcards
High LET
E.g. alpha particles, neutrons, heavy ions
- Direct action (e.g. ionisation causing SSBs and DSBs from the beam) is dominant
- Useful in slow growing tumours
- Higher incidence and severity of late toxicities
- Less split dose recovery and sparing with low dose rate radiation compared to low LET
LET
Average energy transferred per unit of length of the track.
Inversely proportional to the incident energy of the particle. (higher energy = lower LET)
Proportional to the charge of the radiation (higher charge = higher LET)
= dE / dx
dE = energy transferred
dx = distance particle travelled
Low LET
e.g. photons, electrons - LET varies with energy
Indirect action is dominant (approx 66% of cases)
(protons - low LET initially but high at Bragg peak)
Optimum LET
100 kEv/um - optimised to diameter of DNA helix
Highest probability of DSBs with a single charged particle - DSBs are basis of most biological effects
Xrays are sparsely ionising therefore a low probability of a single particle causing a DSB - therefore have a low biological effectiveness
optimal = low energy neutrons/protons/alpha particles
No of SSB & DSB per Gy
Ionising radiation induces approximately 1000 SSB & 40 DSB per Gy.
SSBs potentially mutagenic - may evolve into DSBs if unrepaired. Little biological consequence in terms of cell killing as can repair other strand as template.
DSBS potentially lethal, may induce chromosomal abberrations - most important type of damage for radiotherapy.
Cell death after radiation correlates with unrepaired DSBs.
Damage can occur to sugar phosphate backbone or to bases, or both - cell requires different repair mechanisms for different types of damage.
Chromosome & chromatid aberrations
- Chromatid aberrations - damage to 1 arm of the duplicated chromosome (2 chromatids), occurs after irradiation in G2 (ie after S phase)
2.Chromosome aberrations - single chromatid strand is broken BEFORE duplication in S phase. Leads to chromosome abberation after the next mitosis.
Possible consequences after chromosome broken:
- Broken ends re-join their original positions - no issue
- Fragment of the chromosome maybe lost if there are several DSBs
- Broken ends may attach to incorrect broken ends - lethal or stable.
- Reciprocal translocation - stable and detected long after exposuer
3 lethal chromosomal abberations:
Detected by spectral karyotyping
- Dicentric chromosomes - 2 centromeres (short arm of 2 centromeres detached) - new chromosome containing 2 centromers gets dupliated in S phase -> dicentric chromosome.
- Ring chromosome - both ends are lost from same chromosome early in the cell cycle - chromosome attaches its 2 neds together - RING. Then in S phase chromosome replicates - overlapping ring.
- Anaphase bridge - happens after chromosome is replicated (G2). Both chromatids suffer DSB and sticky ends join together. At anaphase unable to separate the fused arms after centromere breaks - hinders separation of 2 cells - almost always lethal!
Non lethal consequences
- Symmetrical translocations - a break in 2 pre-replication G1 chromosomes - the broken ends are exchanged, can be seen with FISH.
- Small deletions - two breaks in the same arm of the same chromosome, leading to loss of genetic information between the 2 breaks. Can result in carcinogenesis if TS gene is lost.
Summary of chromosome / Chromatid abberrations
- Chromosome abberation
- Chromatid abberation
- Single hit
- Two hit
- ChromoSOME abberation
- dicentric, translocation, ring, inversion - ChromaTID abberation - anaphase bridge
- Single hit - terminal deletion
- Two hit - dicentric, ring, anaphase, translocation, central deletion, inversion
Direct (1:1) relationship between lethal chromosome abberations + cell survival.
Measuring DNA Damage & Repair
SSBSs - alkaline solution (comet) assay - single gel electrophoresis
DSBs:
1. Gamma- H2AX - a subtype of histone ‘h2a’ in response to ionising radiation is rapidly phosphorylated (within 15 mins). A reliable measure to specifically localise DSBs within the nucleus - gamma h2ax fous assay. The most sensitive assay for DSBs. (1 foci = 1 DNA DSB, larger foci = multiple breaks in 1 location).
- pulsed field electrophoresis - negatively charged DNA migrates toward the cathode.
- Neutral comet assay
DNA Damage Response
Sensors = PH2ax, mrn, RPA, RAD 1,9,17; Hus 1, MDC1
Transducers - ATM, ATR
Mediators - CHk1 / 2
Effectors = p53
ATM & ATR (Transducers of DNA)
ATM is transducer of DSBS
ATR is transducer of SSBs
ATM activates p53 & Chk2 - activation of G1 checkpoint (cell cycle arrest at g1 prior to S phase and DNA synthesis - chance for repair and DNA fidelity maintained).
ATR activates CHK1 - activation of G2 checkpoint - cell cycle arrest at G2 prior to M phase and mitosis. Chance for repair and DNA fidelity maintained.
Consequences after Irradiation:
1. Repair
2. Mitotic catastrophe
3. Apoptosis
4. Mutation
5. Carcinogensis
6. Adaptive repsonse
7. Bystander effects
- Repair - cell cycle arrest & repair of DNA damage
- Mitotic catastrophe - cell dies when attempting first or subsequent mitosis, most common cause of death after IR. (result of chromosome abberrations, cells enter cell cycle and die from apoptosis/necrosis or senesscence).
- Apoptosis
- Mutation - chrosomal abberations - genetic instability
- Carcinogenesis - mutation transforms cell phenotype
- Adaptive response - irradiated cell becomes resistant to subsequent irradiation
- Irradiated cell sends signal to neighbouring naive cells to induce genetic damage in them.
Mechanism of Cell death
Depends on cell type and p53 status (P53 prevents apoptosis and stimulates cell cycle arrest and repair)
Proliferating cells - mitotic catastrophe
Cells in interphase - die of apoptosis
Non proliferating (mature) cells - loss of specific function - considered death.
Compare bystander effects with direct effects of radiation
Cells respond to their neighbours being irradiated via Gap junctions and via release of soluble factors, cytokines, ROS, and nitric oxide by the irradiated cells.
Responses include DNA damage, mutations, cells death even without direct irradiation.
