Radiation effect on cell

By Dr. Deepa Gautam

1 yr Resident, Radiotherapy

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Radiation Absorption

• Absorption of energy from radiation in biologic material may lead to:

• Excitation: raising of an electron in an atom to higher level

• Ionization: ejecting one or more electrons from an atom (the radiation is known as ionizing radiation).

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Types of Ionizing Radiation

• Directly Ionizing: when absorbed in material, they directly cause ionization leading to damage. Eg. Electrons, α-particles, β-particles

• Indirectly ionizing: when absorbed in material, they give up their energy to produce fast moving charged particles which produce the damage. eg. Electromagnetic radiation

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Electromagnetic radiation

• Electric and magnetic fields are perpendicular to each other

• Eg. X-rays and γ-rays , occupy the short wavelength end of electromagnetic spectrum

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Action of Radiation

• Direct: when absorbed, radiation directly interacts with targets in the cells and the atoms get ionized or excited.

• Indirect: when absorbed, radiation interacts with other atoms or molecules in the cell particularly water to produce free radicals that produce the damage.

• H20→ H20++e-

• H20++H20→ H30++OH.

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Modes of cell death after irradiation:

• Mitotic Death

• Interphase Death

• Apoptotic Death

• Necrotic Death

• Autophagy

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Mitotic Death

• Cells lethally injured by clinically relevant doses of radiation execute one or more divisions before mitotic death

• Mechanisms :

– Failure of spindle formation in M phase

– Loss of G2 check point

– Improper chromosomal segregation due to damage and loss of genetic material

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Interphase Death

• Occurs in radiosensitive cells

• Occurs within 2-6 hrs of radiation

• Cells dying in interphase cannot contribute to reproductive pool

• Death occurs by rapid apoptosis

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Apoptotic death

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Necrotic Death

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Autophagy

• Cells internalize cellular organelles within the vacuoles and digest them

• Also a type of programmed cell death

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Radiation Injury In Normal Tissue

• Acute Response

• Subacute Response

• Late Response

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Acute response

• Occurs during standard 6-8 wks course

• Depletes stem and progenitor cell pools

• Severity of injury depends upon extent of cellular depletion and length of delay before new functional cells are released

• Severity increases with dose and fractionation decreases severity allowing time for regeneration

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Subacute Response

• Occurs few to several months after irradiation

• Symptoms are usually reversible but sometimes may be severe to cause death

• Mostly occurs during remodelling phase

• Eg. somnolence after brain irradiation, subacutepneumonitis after lung irradiation

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Late Response

• Occurs due to depletion of slowly proliferating cells that are lost at slow rate(eg. Renal tubular epithelium, oligodendrocytes, schwanncells,endothelium,fibroblasts)

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• DNA is the principal target in the cell for biologic effects of radiation.

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DNA

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Effects of radiation on DNA

• Single strand break (SSB)

• Double strand break (DSB)

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SSB

• Repaired readily using opposite strand as a template

• Misrepair may result in mutation

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DSB

• Well separated breaks in two strands repair in similar ways as SSB

• Breaks in two strands opposite one another or separated by only few base pairs may lead to DSB in which the piece of chromatin breaks into two pieces

• Most important lesions in chromosomes produced by radiation

• May result in cell killing, carcinogenesis or mutation

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Radiation induced Chromosomal Aberrations:

• DSBs occur

• Sticky broken ends can join with other sticky end

• Possibilities:

– Rejoin in their original configuration

– Fail to rejoin, give aberrations and deleted in next mitosis

– Broken ends rejoin and form grossly distorted chromosome

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• Aberrations viewed at metaphase of mitosis as:

– Chromosomal aberration

– Chromatid aberration

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Chromosomal aberrations

• cells irradiated in early interphase before chromosomal duplication

• Break occurs in single strand of chromatin

• Chromatin lays down an identical strand with break during synthesis phase

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Chromatid Aberrations

• Cells irradiated after chromosomal duplication

• Break may occur in one of the sister chromatids or in both but not at the same place as chromatids are well separated except in the region of centromere

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Lethal Aberrations:

1. Dicentric chromosome

2. Ring chromosome

3. Anaphase bridge

1 and 2 are chromosome aberrations and 3 is chromatid aberration

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Dicentric chromosome

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Ring chromosome

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Anaphase bridge

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Non-lethal Chromosomal aberrations

•Symmetric Translocations

Break in two pre-replication(G1) chromosomes with broken ends being exchanged

•Small Deletions

Two breaks in the same arm of a chromosome leading to the loss of genetic information between two breaks

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DNA Repair Pathways:

• Base Excision Repair(BER)

• Nucleotide Excision Repair(NER)

• DNA DSB Repair

• Single Strand Annealing

• Cross link Repair

• Mismatch Repair

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Base Excision Repair

•Base damage repaired by this process

•Incorrect base removed by DNA glycosylase /lyase

•Sugar residue removed by Apurinic Endonuclease1

•Correct base replaced by DNA polymerase β

• Sealed by DNA ligaseIII-XRCC1(X-Ray cross complementing factor1)

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BER for multiple nucleotides

•Incorrect bases removed by Apurinic Endonuclease1

•Repair synthesis by complex of RFC/PCNA/DNA polymerase δ/ε

•Unwanted flap removed by FEN1 endonuclease

•Sealed by DNA ligase I

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Nucleotide excision repair

• Removes bulky adducts in DNA like pyrimidinedimers

• Steps:

– Damage recognition

– DNA incisions that bracket the lesion

– Removal of adducts containing region

– Repair synthesis to fill the gap

– DNA ligation

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DNA DSB repair

• Repaired by two processes:

– Homologous Recombination Repair(HRR)

– Nonhomologous End Joining(NHEJ)

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Homologous Recombination Repair

• Occurs in late S/G2 phase

• Undamaged sister chromatid acts as template

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ATM(ataxia telangiectasia mutated) and ATR(AT and Rad3 related)sense the DSB and recruit to the site

H2AX phosphorylated

BRCA1 recruits to the site to regulate the activity of NBS/MRE11/Rad50s protein complex

Unidentified endonucleases and MRE11 resect the DNA

Rad51 binds to 3’ single strand DNA

BRCA2 recruited

Rad51 loaded on RPA coated single strand

Rad52 recruited which protects against exonucleolytic degradation

Rad54 unwind the double stranded molecule

Two invading ends serve as primers for DNA synthesis

Holiday junctions resolved by MMS4 and MUS81 by non crossing over or crossing over

Gap filling of DNA strand

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NHEJ

• Occurs in G1 phase

• Steps:

1. End recognition by Ku heterodimer and DNA dependent protein kinase catalytic subunit

2. End processing by a protein Artemis forming complex with DNA-PKcs and endonuclease activity activated

3. End bridging or fill-in synthesis by DNA polymeraseµ

4. Ligation by XRCC4/DNA ligase IV complex

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Single Strand Annealing

• Plays a transition role between HRR and NHEJ

• Ends of DSB digested by endonuclease(NBS/MRE11/Rad50 complex) until the region of homology are exposed on both ends of the breaks

• Nonhomologous ends are removed and two ends are ligated

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Cross-link Repair

• Cross links occur between DNA-DNA and DNA-proteins

• Nucleotide excision repair and recombinationalpathways combine to repair these cross links

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Mismatch Repair

• Removes base-base and small insertionalmismatches occuring during replication and also during HRR

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Operational Classification of radiation damage:

• Lethal damage: irreversible and irreparable damage that leads to cell death

• Potentially lethal damage: causes cell death under ordinary circumstances but can be modified by postirradiation environmental conditions

• Sublethal damage: repairable in hours under ordinary circumstances unless additional sublethaldamage is added

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References:

• Perez and Brady’s Principles and Practice of Radiation Oncology, 5th Edition

• Radiobiology for the Radiologist by Eric J. Hall and Amato J. Giaccia ,6th Edition

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Thank you

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