4 r’s of radiobiology

4 R’S OF RADIOBIOLOGY

Dr Nanditha Kishore

Biological basis

Cellular targets for radiationBiological factors influencing radio

sensitivityChemical modifiers of radiation

response

DNA damage by ionizing radiation Biological effects of ionizing

radiation are largely the result of DNA damage.

A.DIRECT DAMAGE :in case of high LET radiations such as ALPHA particles,nuetrons

B.INDIRECT DAMAGE:IN case of low LET radiations such as x-rays.

BIOLOGICAL BASIS

A consideration of biological effects of radiation should begin with three aspects

1.Types of DNA breaks

2.Radiosensitivity in cell cycle

3.Advantages of dose fractionation

TYPES OF DNA DAMAGE DNA is a large molecule with a well

known double helical structure.

The “backbone” of each strand consists of alternating sugar and phosphate groups.

Attached to this backbone are four bases, the sequence of which specifies the genetic code.

Pyrimidines= thymine and cytosine. Purines= adenine and guanine

Do DOSE=A dose of radiation that induces an average of one lethal event per cell leaves 37% still viable .

For mammalian cells X-RAY Do dose lies between 1-2 Gy.

Number of DNA lesions per cell Base damage=>1000 SSB=1000 DSB=40

DNA BREAKS

2.Radiosensitivity in cell cycle

Biological basis of survival curve

Advantages of Fractionation

The Advantages Of Dose Fractionation Include

Reduction in the number of hypoxic cells through cell killing and reoxygenation.

Reduction in the absolute number of clonogenic tumor cells by the preceding fractions with the killing of the better oxygenated cells.

Blood vessels compressed by a growing cancer are decompressed secondary to tumor regression.

Fractionation exploits the difference in recovery rate between normal, acute, and late-reacting tissues and tumors.

Radiation-induced redistribution of cells within the cell cycle tends to sensitize rapidly proliferating cells as they move into the more sensitive phases of the cell cycle.

The acute normal tissue toxicity of single radiation doses can be decreased with fractionation.

Thus, patients' tolerance of radiotherapy will improve with fractionated irradiation.

Biological factors influencing radio sensitivity Efficacy of fractionation can be related to the

“Four Rs” of Radiobiology:

REPAIR OF SUBLETHAL DAMAGE

REASSORTMENT OF CELLS WITHIN THE CELL CYCLE

REPOPULATION

REOXYGENATION

OPERATIONAL CLASSIFICATIONS OF RADIATION DAMAGE (1) LETHAL DAMAGE= which is irreversible

and irreparable and leads irrevocably to cell death;

(2) POTENTIALLY LETHAL DAMAGE (PLD)= The component of radiation damage that can be modified by postirradiation environmental conditions.

(3) SUBLETHAL DAMAGE (SLD)=which under

normal circumstances can be repaired in hours unless additional sublethal damage is added

TYPES OF DNA REPAIR Mammalian cells have developed

specialized pathways to sense, respond to, and repair these different types of damage.

Different repair pathways are used to repair DNA damage, depending on the stage of the cell cycle.

The stability of repair pathway determine the radiosensitivity of cell cycle phases.

PATHWAYS OF DNA REPAIR Base Excision Repair (BER)

Nucleotide Excision Repair (NER)

DNA Double-Strand Break Repair 1. Nonhomologous End Joining

(NHEJ) 2. Homologous Recombination

Repair (HRR)

OTHERS Single-Strand Annealing (SSA)

Cross-Link Repair

Mismatch Repair

Base Excision Repair (BER)Singlebase mutation that is first removed

by a glycosylase/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.

The process can be subdivided into pathways

1.Globel genome repair(GER) 2.Transcription coupled repair(TER)

The mechanism differs only in the detection of lesion

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.

Defective NER increases sensitivity to UV-induced DNA damage and anticancer agents such as alkylating agents that induce bulky adducts.

Germline mutations in NER genes lead to human DNA repair deficiency disorders such as xeroderma pigmentosum

REPAIR OF DNA DSB’S

Nonhomologous 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) Homologous recombination repair

(HRR) is a High-fidelity mechanism of repairing DNA DSBs.

Its function primarly in late S/G2 is to repair and restore the functionality of replication forks with DNA double-strand breaks.

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

HEREDITARY SYNDROMES THAT AFFECT RADIOSENSITIVITY Ataxia-Telangiectasia (AT)

Ataxia-Telangiectasia-Like Disorder (ATLD)

Nijmegen Breakage Syndrome (NBS)

Fanconi Anemia (FA)

Split dose repair split dose repair (SDR) that manifests its

importance during fractionated radiotherapy.

SDR describes the increased survival found if a dose of radiation is split into two fractions compared to the same dose administered in one fraction.

Molecular mechanisms for SDR are unknown, experimental evidence suggests that this repair is due to DNA double-strand break rejoining.

Elkind et al study on SLD

INCUBATED AT NORMAL GROWTH CONDITIONS

This simple experiment, performed in vitro, illustrates three of the “four Rs” of radiobiology: repair, reassortment, and repopulation.

Reassortment and repopulation appear to have more protracted kinetics in normal tissues than rapidly proliferating tumor cells.

RE-ASSORTMENT Cells change in their radiosensitivity

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 synchronised.

If allowed time between fractions they become SELF SENSITISED.

This phenomenonof SELF SENSITIZATION due to movement through cell cycle is called RE-DISTRIBUTION or RE-ASSORTMENT.

This will occur only in a proliferating cell population.

Thus therapeutic ratio can be enhanced .

The differential is greater the smaller the dose per fraction and proportional to number of fractions.

RELATION TO NORMAL TISSUES

Dose rate effect and inverse 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

SURVIVAL CURVES AT WIDE DOSE RATES AS Dose rate is reduced survival

curve becomes shallower and shoulder tends to disappear.

This is most dramatic between o.o1 and 1Gy /min.

Magnitude of dose rate effect varies among types of cells

HE LA CELLSHAMSTER CELLS

Cell lines from human origin tends to fan out at LDR

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.

This is due to phenomenon of RE-DISTRIBUTION.

SUMMARY OF DOSE RATE EFFECT

RE-POPULATION It occurs as a homeostatic response

to cell depletion caused by treatment.

It is mainly observed in 1.Acute –Responding normal tissurs 2.Tumors With high rate of cell

production. The cell loss after each fraction of

radiation induces compensatory cell regeneration the extent of which determines tissue tolerance.

POTENTIAL DOUBLING TIME It Is The Pre Irradiation Proliferative

Activity measured by time required for the number of clonogenic cells to double assuming cell loss factor as zero.

Tpot = c Ts/LI

GROWTH FRACTION=It increases following cytoreductive therapies and leads to ACCELERATED RE-GROWTH.

It is assessed by increase in dose required for tumor control as duration of treatment increases.

OR For a constant dose decrease in

tumor control rate as treatment time is extended

Supporting observations1.Time to recurrence One tumor cell must undergo 30

doublings to become detectable as recurrence.

Most recurrences in HEAD & NECK cancer occur within 12 months after radiation therapy.

Median doubling time at presentation is usually 2 months.

2.Split course treatment These Schedules resulted in lower local control

rates .

3.Protracted treatment It resulted in decreased rate of locoregional

control and led to worse outcomes in several analyses.

For treatment durations of 30-55 days EACH 1 DAY EXTENSION=O.6 Gy INCREASE IN

TOTAL DOSE to achieve constant rate of tumor control

4.Accelerated treatment If accelerated tumor growth

contributes to treatment failure, acceleration of standard treatment may benefit some tumors.

In nonrandomized studies this improved the local control in inflammatory breast cancer , melanoma metastases to brain.

Randomized studies of accelerated treatment of head and neck cancer validated the benefit.

TCD50 analysis.

These values are independent of treatment duration up to about 28 days, after which they increase rapidly (consistent with 0.6 Gy/day).

Head and neck SCCs exhibit a lag period of 3 to 4 weeks before beginning to repopulate with an average doubling time of 3 to 4 days.

Re population in oro pharyngeal cancers

Influence of regeneration

Normal tissues Time to onset of repopulation after

irradiation and rate at which it proceeds vary with the tissue.

In humans tissue turn over kinetics are slower than mice.

High initial doses shorten the LAG PERIOD.

It confers benefit by reducing toxicity in acute responding tissues.

Growth factors may be useful in protecting normal tissues from irradiation by shortening the apparent lag phase and accelerating recovery in irradiated tissues.

Hematopoietic growth factors Keratinocytic growth factors

Tumor tissues Like acute responding normal tissues

tumors accelerate their growth in response to injury.

ACCELERATED REPOPULATION It is division of surviving clonagens of

cells in a tumor at a faster rate than before being triggered by any cytotoxic agent including radiation.

Accelerated repopulation

PRACTICALS IMPLICATIONS OF RE-POPULATION1.Protracting treatment longer than

necessary will likely be a disadvantage. using 1.8 Gy rather than 2 Gy fractions

given five times per week extends overall treatment time by about 10% .

RESERVED FOR SITUATIONS IN WHICH 1.Acute responses limiting dose

accumilation. 2.Ih homogenicities in dose distribution

2. If a break in treatment is necessary because of acute toxicity, it should be kept as short as is tolerable.

3.Planned split-course therapy is inadvisable unless it is part of an accelerated treatment protocol that ultimately shortens the overall treatment duration .

4.Breaks in therapy for nonmedical reasons (machine breakdown, holidays) may merit catch up treatments in patients being treated for cure

“ Rapidly growing tumors must be treated rapidly”

Treatment should never be unnecessarily protracted because it is difficult to predict the accelerated repopulation response of individual tumors.

Re-oxygenation

Effect Of Oxygen On Radiosensitivity The oxygen effect was observed as early as 1912

in Germany by Swartz. In England in the 1930s, Mottram explored the question of oxygen in detail.

oxygen enhancement ratio (OER).

doses administered under hypoxic condition

doses administered under aerated conditions

to achieve the same biological effect.

It varies with 1.Type of radiation

2.Dose assay

Variation With Type Of Rays

Variation With Dose Assay

MECHANISM OF THE OXYGEN EFFECT

The Oxygen Fixation Hypothesis Absorption of radiation fast charged particles

number of ion pairs

free radicals R+DNA=R.+ O2 =R02(FIXATION OF DAMAGE)

break chemical bonds

chemical changes

Final expression of biologic damage

FRACTIONATION TO TACKLE HYPOXIA

The oxygen status of cells in a tumor is not static; it is dynamic and constantly changes.

Proportion of hypoxic cells in the tumor is about the same at the end of a fractionated radiotherapy regimen as in the untreated tumor.

During the course of the treatment hypoxic cells become oxygenated.

This phenomenon, by which hypoxic cells become oxygenated after a dose of radiation, is termed reoxygenation..

TUMOR REOXYGENATION

Time sequence of RE-OXYGENATION(mouse sarcoma cells)

RATE and EXTENT of Re-oxygenation varies with type of tumors.

If this is appropriately studied it can be used in designing fractionatation shedules.

Since pattern is not known for many human tumors it can considered that doses on the order of 60 Gy (6,000 rad) given in 30 treatments argues strongly in favor of reoxygenation.

Re –oxygenation in various tumors

Mechanism of Re-Oxygenation Opening up of blood vessels Decreased diffusion distance(70um-

150um) Revascularization of tumor

Chemotherapy Induced Radiosensitization

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