1) The four Rs of radiobiology are repair, re-assortment, repopulation, and re-oxygenation. They influence how tumors and normal tissues respond to fractionated radiation treatment. 2) When radiation is delivered in two fractions separated by time, cell survival increases due to repair of sublethal damage between fractions. The increase peaks at 2-3 hours and then levels off due to repopulation. 3) Lowering the radiation dose rate generally decreases biological effects because it allows more time for repair of sublethal damage.

1. 4R's of Radiobiology and
dose rate effect
Dr. Laxman pandey
JR3
Department of radiotherapy

2. Definition
The most important biological factors influencing the responses of tumours and
normal tissues to fractionated treatment are often called the “four Rs”:
● Repair of sub lethal damage
● Re assortment of cells within the cycle
● Repopulation
● Re oxygenation

3. Radiation Induced DNA Damage
Critical target – DNA
Radiation when absorbed in
biological material may damage DNA
by any of following
Direct action –
Radiation interacts with critical
target.
Atoms of target get ionized & lead to
biological damage
Indirect action-
Secondary e- interacts with e.g.
water molecule to produce free
radicals which damage DNA &
produce biological changes.

4. Types of Damage:-
● Lethal—irreversible, irreparable, leads to cell death
● Sub lethal (SLD)—repaired in hours; if a second dose is given, can interact with
more damage to create lethal damage; represents shoulder on cell survival curve.
● Potentially Lethal Damage (PLD)—can be modified by the post-irradiation
environment.

5. 1.REPAIR
PATHWAYSOFDNAREPAIR
BaseExcisionRepair(BER)
NucleotideExcisionRepair(NER)
DNADouble-StrandBreakRepair
1.NonhomologousEndJoining(NHEJ)
2.HomologousRecombinationRepair(HRR)
OTHERS
 Single-StrandAnnealing(SSA)
Cross-LinkRepair
MismatchRepair

6. Base Excision Repair (BER)
Single base mutation that is first
removed by a glycosylate/DNA
lyase .
Removal of the sugar residue by an
AP endonuclease
Replacement with the correct
nucleotide by DNA polymerase
Completed by DNA ligase III
XRCC1-mediated ligation

7. NUCLEOTIDE EXCISION REPAIR
Nucleotide excision repair removes bulky adducts in the DNA such as pyrimidine
dimers.
STEPS:-
(1) damage recognition,
(2) DNA incisions that bracket the
lesion, usually between 24 and 32
nucleotides in length
(3) removal of the region containing
the adducts,
(4) repair synthesis to fill in the gap
region
(5) DNA ligation

8. REPAIR OF DNA DOUBLE STRAND BREAK

9. Non homologous End Joining (NHEJ)
Steps
(1) end recognition(Ku hetero dimer
and DNA PKcs)
(2) end processing(Artemis protein)
(3) fill-in synthesis or end bridging
(DNA polymerase µ)
(4) ligation (XRCC4/DNA ligase IV
complex )

10. Homologous Recombination Repair (HRR)
HRR is a High-fidelity mechanism of
repairing DNA DSBs.
Its function primarily in late S/G2 .
HRR requires physical contact with an
undamaged chromatid or chromosome
(to serve as a template) for repair to
occur.

11. STEPS
1.Recognition of damage(ATM protein
kinase)
2.Recruitment of proteins(H2AX, BRCA1,
SMC1, Mre11, Rad50, and Nbs1)
3.Resection of DNA(MRE11 )
4.Strand exchange(BRCA2 and RAD51)
5.DNA synthesis(Using undamaged strand as
primer)
6.Resolution of HOLIDAY junctions.(MMS4
and MUS81 by non-crossing over)
7.Gap filling
8.ligation

12. Sublethal Damage (SLD) Repair
The repair of sub lethal damage reflects
the repair and re joining of double-
strand breaks before they can interact to
form lethal lesion.
If a dose is split into two parts separated
by a time interval, some of the double-
strand breaks produced by the first dose
are re joined and repaired before the
second dose and more cells survive.
Survival of Chinese hamster cells exposed to
two fractions of x-rays and incubated at 24c
for various time intervals between the two
exposures

13. INCUBATED AT NORMAL GROWTH CONDITIONS
This simple experiment, performed in vitro,
illustrates three of the “four Rs” of
radiobiology: repair, re assortment, and
repopulation.
Re assortment and repopulation appear to
have more protracted kinetics in normal
tissues than rapidly proliferating tumor cells
Survival of Chinese hamster cells exposed to two fractions of x-rays
and incubated at 37° C for various time intervals between the two
doses. The survivors of the first dose are predominantly in a resistant
phase of the cycle (late S). If the interval between doses is about 6
hours, these resistant cells have moved to the G2M phase, which is
sensitive.

14. A: If the dose is delivered in two fractions separated by a time interval, there is an increase in cell survival
because the shoulder of the curve must be expressed each time. B: The fraction of cells surviving a split
dose increases as the time interval between the two dose fractions increases. As the time interval
increases from 0 to 2 hours, the increase in survival results from the repair of sublethal damage. In cells
with a long cell cycle or that are out of cycle, there is no further increase in cell survival by separating the
dose by more than 2 or 3 hours. In a rapidly dividing cell population, there is a dip in cell survival caused by
reassortment.
Summary of the repair of sub lethal damage as evidenced by a split
dose experiment.

15. 2.RE-ASSORTMENT
Cells change in their radio sensitivity as they traverse the cell cycle.
After exposure of asynchronous population of cells to radiation those in the
sensitive phase are killed thus becomes partly synchronized.
If allowed time between fractions they become SELF SENSITISED.
This phenomenon of SELF SENSITIZATION due to movement through cell
cycle is called RE-DISTRIBUTION or RE-ASSORTMENT.
“Sensitization due to re-assortment” causes therapeutic gain.

16. 3.Re-population
In b/w dose fractions normal cells as well as tumor cells repopulate.
So longer a radiotherapy course lasts, more difficult it becomes to
control tumor & may be detrimental.
But acutely responding normal tissue need to repopulate during course
of radiotherapy .
Thus fractionation must be controlled so as not to allow too much time
for excessive repopulation of tumor cells at the same time not treating
so fast that acute tolerance is exceeded

17. ACCELERATED REPOPULATION
In normal tissues Repopulation occurs in different speeds depending on the
tissue.
Early responding tissues begin repopulation at about 4 weeks. By increasing
treatment time over this amount, it is possible to reduce early toxicity in that
tissue.
Late responding tissues only begin repopulation after a conventional course of
radiation has been completed, and therefore repopulation has minimal effect on
these tissues.
Treatment with any cytotoxic agent, including radiation, can trigger surviving
cells (clonogens) in a tumor to divide faster than before. This is known as
accelerated repopulation.

18. PRACTICALS IMPLICATIONS
It may be better to delay initiation of treatment than to introduce delays during
treatment.
Protracting treatment longer than necessary will likely be a disadvantage.
e.g. using 1.8 Gy rather than 2 Gy fractions given five times per week extends overall
treatment time by about 10% .
If overall treatment time is too long, the effectiveness of later dose fractions is
compromised because the surviving clonogens in the tumor have been triggered into
rapid repopulation.
If a break in treatment is necessary because of acute toxicity, it should be kept as
short as is tolerable.
Planned split-course therapy is inadvisable unless it is part of an accelerated
treatment protocol that ultimately shortens the overall treatment duration .

19. 4.REOXYGENATION
Phenomenon by which hypoxic cells become
oxygenated after a dose of radiation is termed re
oxygenation.
A modest dose of x-rays to a mixed population of
aerated and hypoxic cells results in significant killing of
aerated cells, but little killing of hypoxic cells.
Consequently, the viable cell population immediately
after irradiation is dominated by hypoxic cells.
If sufficient time is allowed before the next radiation
dose, the process of re oxygenation restores the
proportion of hypoxic cells to about 15%.
If this process is repeated many times, the tumor cell
population is depleted, despite the resistance of
hypoxic cells to killing by x-rays.

20. Mechanism of Re-Oxygenation
1.Opening up of blood vessels, (fast component)
2.Decreased diffusion distance(70um-150um), (slow component)
3.Revascularization of tumor

21. SLOW COMPONENT
TAKES PLACE OVER A PERIOD OF DAYS IN CHRONICALLY HYPOXIC CELLS
After a dose of radiation
Tumor cells killed and removed from population
tumor shrinks in size and restructuring or a revascularization of
the tumor occurs
surviving cells previously beyond the range of oxygen diffusion
become closer to a blood supply and so re oxygenate.

22. FAST COMPONENT
Complete within hours
Caused by the re oxygenation of acutely hypoxic cells.
Those cells that were hypoxic at the time of irradiation because they were in
regions in which a blood vessel was temporarily closed quickly re oxygenate
when that vessel is reopened.

23. Importance of Re oxygenation in RT
If all the human tumors re oxygenate rapidly , use of a multifraction course
of radiotherapy, extending over a period of time, can deal effectively with
any hypoxic cells in human tumors.
Making optimal choice of fractionation, demands a detailed knowledge of
the time course of re oxygenation in the particular tumor to be irradiated.
Unfortunately, this information is available for only a few animal tumor and
no information at present for human tumor. Indeed, in humans it is not
known with certainty whether any or all tumors re oxygenated

24. Dose rate effect
Dose rate is one of the principal factors that determine the biological
consequences of absorbed radiation.
As dose rate
exposure time increases
Biological effect generally
This Is due to SUB LETHAL DAMAGE REPAIR

25. IDEALIZED FRACTIONATION EXPERIMENT
Curve A is the survival curve for single
acute exposures of x-rays.
Curve F is obtained, if each dose is given as
a series of small fractions of size D1 with
an interval between fractions sufficient for
repair of sub lethal damage.
Multiple small fractions approximate to a
continuous exposure to a low dose rate.

26. The survival curves fan out
at LDR because in addition to a range
of inherent radio sensitivities (evident
at HDR), there is also a range of repair
times of sub lethal damage.
Cell lines from human origin tends to
fan out at LDR
Dose survival curves for 40 different cell lines of human origin at high dose rate
and low dose rate

27. INVERSE DOSE RATE EFFECT
IN converse with usual phenomenon,
increased cell killing is seen with decrease in
dose rate called the INVERSE DOSE RATE
EFFECT.
The inverse dose-rate effect. A range of dose rates
can be found for HeLa cells such that lowering the
dose rate leads to more cell killing. At 1.54 Gy/h, cells
are “frozen” in the various phases of the cycle and do
not progress. As the dose rate is dropped to 0.37
Gy/h, cells progress to a block in G2, a radiosensitive
phase of the cycle.

28. SUMMARY OF DOSE RATE EFFECT
The dose-rate effect resulting from repair
of sub lethal damage, redistribution in the
cycle, and cell proliferation.
The dose-response curve for acute
exposures is characterized by a broad
initial shoulder.
As the dose rate is reduced, the survival
curve becomes progressively more shallow
as more and more sub lethal damage is
repaired, but cells are “ frozen” in their
positions in the cycle and do not progress.

29. As the dose rate is lowered further and for a limited range of dose rates, the
survival curve steepens again because cells can progress through the cycle to
pile up at a block in G2, a radiosensitive phase, but still cannot divide.
A further lowering of dose rate below this critical dose rate allows cells to
escape the G2 block and divide; cell proliferation then may occur during the
protracted exposure, and survival curves become shallower as cell birth from
mitosis offsets cell killing from the irradiation.