4R's of Radiobiology and dose rate effect

Dr. Laxman pandey JR3Department of radiotherapy

Definition

The most important biological factors influencing the responses of tumours andnormal 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

Radiation Induced DNA Damage

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

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.

1.REPAIRPATHWAYS OF DNA REPAIRBase Excision Repair (BER)

Nucleotide Excision Repair (NER)

DNA Double-Strand Break Repair 1. Non homologous End Joining (NHEJ) 2. Homologous Recombination Repair (HRR)OTHERS Single-Strand Annealing (SSA)

Cross-Link Repair

Mismatch Repair

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

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

REPAIR OF DNA DOUBLE STRAND BREAK

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 )

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.

STEPS1.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

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

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 cellsSurvival 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 resistantphase 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.

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.

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.

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

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.

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 .

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.

Mechanism of Re-Oxygenation

1.Opening up of blood vessels, (fast component)

2.Decreased diffusion distance(70um-150um), (slow component)

3.Revascularization of tumor

SLOW COMPONENTTAKES 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.

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.

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

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

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.

The survival curves fan outat LDR because in addition to a rangeof inherent radio sensitivities (evidentat HDR), there is also a range of repairtimes 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

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.

SUMMARY OF DOSE RATE EFFECT

The dose-rate effect resulting from repair of sub lethal damage, redistribution in thecycle, 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.

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.