PCB675 Week 6 Cellular Effects of Ionising Radiation

What is Radiobiology, define the 2 levels.

Study of radiation effect at cellular and molecular levels

Cellular level: the basic functional unit of human

Molecular level: such as water, protein, carbohydrates, and DNA

What happens when cell is damaged? (Hint: 3 outcomes)

1. Injured cells repair themselves through defence mechanisms, results in no residual damage

2. The cell dies and is replaced

3. Cell repairs itself improperly, results in a viable but modified cell.

How radiation affects cells? (3 Steps) 1. energy deposition 2. effect of energy deposition 3. chemical bond

Irradiation of biological material results in a random and uneven distribution of energy deposition in tissues and cells.

Much of the deposited energy causes excitation and ionisation.

Random distribution of chemical bond breakage is responsible for biological effects e.g. molecular change

Effects of ionising radiation at various levels

Molecular - DNA

Subcellular - Cell membranes and chromosomes

Cellular - inhibition of cell division

Tissue/Organ - Radiation sickness to gastrointestinal (GI), bone marrow (haematopoietic), and CNS

Whole body - death, cancer

Population - genetic

Biologic Factors - types of cell damage (examples)

Damage to nucleus and DNA

Damage to cell membrane (ion and nutrient leakage)

Damage to mitochondria in the cell’s cytoplasm (food supply of cell affected)

Damage to lysosomes (Causes release of enzyme which results in the cell digesting it’s own internal structures)

DNA damage examples

Base damages

Cross links between different DNA strands

Protein cross link

Intercalation

Point lesions

Single and double strand breaks

DNA damage outcome and reversibility

Can be reversible, less likely when more DNA damage is experienced

The outcome can be cell death, malignant disease, genetic damage

DNA damage - Double strand breakage

A break in both strands of DNA

While lesions (breaks or changes in chemical structure) can travel a few base pairs along the DNA 2 ionisation events are still required near each other, and that possibility is dependent on radiation type and dose.

number of single strand breaks and base damages per unit radiation is typically nearly 2 orders of magntude larger than the number of double strand breaks

number of double strand breaks correlates best with cell kill, therefore:

It is generally assumed that double strand breaks are the most significant lesion for cell kill

Direct and indirect effects of IR

Direct - ionising effect occurs on radiosensitive moelcule (DNA)

Indirect - Initial ionising event occurs on any other molecule, and energy is transferred to the DNA

What does Radiolysis of water result in the production of?

Free radicals

Radiolysis of water - principal damaging products after radiolysis of water are formation of…

OH* + OH* -> H2O2 (Hydrogen Peroxide Formation)

H* + O2 → HO2* (Hydroperoxyl formation)

Radiolysis of water - life time of a radical

The longer the life time of a radical the further it can travel which makes H2O2 (hydrogen peroxide) an important radiation product in radiobiology

Radiolysis of water - damaging effects of free radicals and relation with oxygen

Damaging effects of free radicals is enhanced by oxygen! (reduces the probability of the radicals converting back to simple water or hydrogen).

Indirect effect of IR - What happens if the free radical formation occur close to the DNA?

The radicals can diffuse to react with the DNA, causing radiation damage (indirect effect)

What effect is the most common to cause damage in low LET (x-rays, gamma, electrons) IR? how common?

Indirect effect, accounts or 2/3 of damage on low LET IR, leaving direct effect causing 1/3 damage .

Why is indirect effect so damaging?

Due to cells being made up of mostly water, the IR is most likely to reach water molecules which is where radiolysis occurs via the indirect effect.

Surviving fraction

expression of the magnitude of the effect of a given dose of radiation on cell reproductive capacity.

surviving fraction formula

S=Number of cells surviving a given dose/number of cells in the original unexposed sample. = N/N_o

Factors affecting biological radiosensitivity (4 physical)

1. Radiation dose

2. Dose rate, quantity of dose per unit of time

3. Type of radiation: LET and Relative Biologic effectiveness (RBE)

4. Protraction and fractionation

Factors affecting biological radiosensitivity (4 biological)

1. Chemical agents

2. Cell type

3. Phase of cell cycle, age

4. TIme between fractions / Recovery effect

Physical factor - Dose Rate explained

For the same total dose, higher dose rates are more effective than low dose rates due to the lowered potential for repair of radiation damage

Physical factors - Type of Radiation

High LET radiation such as neutrons and protons are more effective at cell killing than low LET radiation

Physical factors - Type of Radiation LET explained, Unit of LET, Diagnostic x-ray LET

LET is a measure of the rate at which energy is transferred from ionising radiation to soft tissue.

Unit - keV/µm

LET of diagnostic x-ray is approximately 3keV/µm

Physical factors - Type of Radiation (Relative Biologic Effectiveness)

RBE (relative biological effectiveness) is another method of measuring relative effectiveness of different types of radiation in producing damage - aka tissue weighting factor

RBE formula

RBE = Dose of standard radiation (250 keV x-rays) necessary to produce a given effect/Dose of another radiation source needed to produce the same biologic response

RBE vs LET for

Physical factors - Protraction and Fractionation explained with examples

Protraction: the dose is delivered continuously but at a lower dose rate, allowing time for cell repair and tissue recovery.

Example: 2Gy/min is lethal for a mouse, but it survives 10mGy/hr for a total of 600hrs.

Fractionation: the dose is delivered in a number of separate fractions over a long time. Cell repair and recovery occur between doses. This is used routinely in radiation oncology.

Example: a mouse survives a 2Gy/min dose rate in 12 equal fractions of 500mGy), each separated by 24hrs.

Surviving fraction - Single dose

Single dose: Initial “shoulder” on the curve indicating accumulation of sublethal damage, followed by almost exponential decrease in surviving fraction.

Surviving fraction - fractionated dose

repopulation and recovery occur between multiple doses – not necessarily at a constant rate. Surviving fraction in fractionated dose is higher than receiving a single dose of the same magnitude.

Biological factors - Sensitisers (oxygen)

Tissues/cells are more sensitive in the presence of oxygen (aerobic state) than in hypoxic (low oxygen)

Useful knowledge in Radiotherapy since tumors have areas of hypoxic cells which are difficult to kill with low LET radiation.

Biological factors - Sensitisers (oxygen) formula

The oxygen enhancement ratio is the ratio of doses which produce the same level of biological effect (e.g. level of survival) in hypoxic compared with oxygenated conditions:

OER = Dose necessary in hypoxic conditions/ Dose necessary in aerobic conditions

Biological factors - Sensitisers (oxygen) The OER is LET dependen… (3 things)

Highest for low LET radiation (max of 3)

OER of 2.5 for diagnostic radiation

Decreases to about 1 for high-LET radiation

Biological factors - Sensitisers (oxygen) 2 types of hypoxia as a result of tumour growth

acute hypoxia resulting from temporary closing/blockage of blood vessels

Chronic hypoxia is a result of long periods of closing/blockage of blood vessels, as some cells are too far from capillary for oxygen to diffuse

tumour shrinkage linking hypoxia and oxygenated cells

Fractionated dose kills some hypoxic cells but more oxygenated cells near the capillaries due to them being more sensitive.

Tumor shrinks towards the capillaries and previously hypoxic cells become better oxygenated.

Process is repeated and tumour shrinks

High LET radiation in relation to hypoxic and oxygenated cell survival

High LET radiation shows similar/equal damage between oxygenated and hypoxic tumour cells

Biological factors - Cell type

Radiosensitivity is greater for cells that:

1. High mitotic rate

2. Long mitotic future

3. Undifferentiated (haven’t formed into final cell type yet)

Biological factors - Phase of the cell cycle

Cells exposed in mitosis and the gap (G2) between preparatory stage for synthesis and mitosis are most sensitive

Less sensitive during late DNA synthesis (S), survival curves show broader shoulder = more chance to repair

Radiosensitivity with age

radiosensitivity decreases with age, which is why radiation safety for children and pregnant women is considered

Biological Factors - Time between fractions, Define the recovery effect of fractionated dose (aka time between fractions).

If radiation is insufficient to kill cell before next division, cell will recover from IR.

Biological factors - cell type (Target theory)

Cell death occurs after radiation exposure and when the “target molecule” is inactivated.

DNA is the principal radiation-sensitive molecule (target molecule)

Mathematical description of target theory (3)

1. Single target, Single Hit model

2. Multitarget, Single hit model

3. Linear-Quadratic model

What happens when radiation damage is inflicted on a molecule which is in abundance within a cell?

May not result in noticeable cell injury as similar molecules are still available to support the cell

What happens when radiation damage is inflicted on a molecule which is NOT in abundance within a cell?

Loss of function as no similar substitutes can carry on the function of the particular cell.

Single-Target, single hit model explained

Start with 100 cells and ascribe a random hit pattern: o After one hit, one of the 100 cells has been hit and killed. o At the second hit, there’s a 99% chance that it will hit a different cell to the first hit and a 1% chance that it will re-hit the first cell. Note: All hits per target in excess of one, represents wasted radiation dose, as the cell has already been killed by the 1st hit). o At the third hit, there’s a 98% chance that it will hit a new cell, and a 2% chance that it will hit a cell that’s already been hit (from hit 1, and hit 2). o As the number of hits increases, it will become more probable that a given cell will be hit twice or more.

Poisson distribution - single target single hit model

when number of hits = number of cells, 63% of cells will be hut at least once, and 37% survive

Calculation of surviving fraction formula

What does a high and low D37 value indicate?

High D37 = low radiosensitivity, low D37=high radiosensitivity

Multitarget, single hit model

The model assumes that within each cell there are multiple targets all of which must be hit once to cause reproductive cell death.

Multi target, single hit model formula

Multitarget, single hit model - explained (Dq, D0, n, shoulder)

At low doses (i.e. the first few hits), there is nearly 100% survival (there is a threshold).

As the D increases, fewer cells will survive (all targets have been hit).

A high D, all cells will be hit at least once on each of the targets and it starts to look like the single hit model - which accounts for the bend (shoulder) in the curve.

D0 (Mean Lethal Dose ) = The dose required to reduce the survival fraction to 37% , is the slope of the straight line portion of the graph.

DQ (Threshold Dose) = a measure of the width of the shoulder of the curve related to the capacity of the cell to recover from the sublethal damage.

Linear Quadratic Model

This is currently the preferred model and assumes that there are two components to cell killing:

One due to single event killing (e.g. double strand break caused by a single ionising particle). The probability of NO damage is e-αD

The other one due to two event killing (e.g. double strand break caused by two separate ionising particles). The probability of NO damage is e-βD 2

Hence: S = e-αD - β D 2 (where α and β are constants)

This relationship has an initial non zero slope which is in agreement with experiment but results in a continuously ‘bending’ curve which may not be the case.