# Radiation Biology for Physical Scientists/Print version

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# Radiation Interaction - Physical and Chemical Events

Radiation Sensitivity of Cells →

## Physical Events
### Energy Deposition in Biological Materials

The energy from radiation needs to be deposited into the cells of biological material before it can produce a biological effect. Charged particle radiation such as alpha particles, electrons and protons directly transfer their energy to other charged particles in cells through Coulomb Interaction. However, uncharged radiation such as x-rays, gamma rays and neutrons cannot directly transfer their energy. Instead, they indirectly transfer all (Photoelectic Effect) or a portion of their energy (Compton Effect) to charged particles in the cells and these charged particles transfer their energy to the biological material through Coulomb Interaction. The units for the energy deposited is Joules per kilogram which is given a special name of Gray in honour of English scientist Louis Harold Gray.

### Excitation and Ionization

The energy deposition can lead to two different events that are dependent on the amount of energy absorbed by the cells.
One type of event is called excitation where the orbital electrons of atoms in the cells get enough energy to make an atomic transit from the ground state to a higher energy level, without leaving the atom. This type of event will not be the focus of this book. The second type of event is called ionization which happens when one or more orbital electrons from an atom in the cell has enough energy to leave the atom leading to ion pairs. Ionization radiation can be further reclassified as directly ionizing and indirectly ionizing depending on the source of the energy.

### Ionization Density

Ionization from radiation in biological material leads to a random and uneven distribution of deposited energy in cells. The spatial distribution of the energy imparted by the charged particle is quantified by the Linear Energy Transfer (LET) metric. It is the quotient of the average energy imparted and the distance traversed by the radiation with units of keV/μm.
Radiation can be reclassified into low LET or high LET radiation based on their LET value. The demarcation value between low and high LET is about 10 keV/μm.
| Low LET Radiation | High LET Radiation |
| X ray | Alpha Particle |
| Gamma ray | Neutron |
| | Proton |

### Direct and Indirect Action

Cells contain organic compounds (proteins, carbohydrates, nucleic acids and lipids), as well as, inorganic compounds (minerals) dissolved or suspended in water. The critical target within the cell for damage from radiation is DNA; however damage to other sites in the cell may lead to cell death. The biological effect of radiation can be classified based on the atoms or or molecules in the cell that are ionized by radiation. Direct Action of radiation refers to the ionization of critical target atoms or molecules in the cell such as DNA. Indirect Action refers to the ionization of atoms of molecules in a cell other than target atoms and molecules. In biological material, the ionization of water (Radiolysis) is important since more than 80% of a cell's mass is water.
Direct Action is the dominant process in the interaction of high LET particles because it results in a denser column of radiation that is more likely to interact directly with DNA. Sparsely ionizing radiation such as low LET particles dominantly interact by indirect action.

## Chemical Events

The average energy dissipated in an ionization event is 33eV which is more than enough to break chemical bonds in the atoms and molecules of cells. The typical energy needed to break the bond in the DNA bases is 9eV. Breaking these chemical bonds causes a chain of chemical events that produces free radicals leading to biological damage. A radical is an atom or molecule that possesses an unpaired electron in its outer shell which usually makes it highly chemically reactive. A free radical is a radical which is able to diffuse from the site it was produced prior to interacting with another molecule.

### Direct Action

Radiation ionizes a target molecule and forms a positively charged radical on the molecule. The letter R is a placeholder for a hydrocarbon group side chain.
RH + radiation → RH•$^+$ + e$^-$
The RH•$^+$ cation radical decomposes into a sugar or base radical and hydrogen cation.
RH•$^+$ + e$^-$ → R• + H$^+$
Chemical reactions with this radical leads to breaks in one or both strands of the DNA helix. These single and double strand breaks will be discussed further in the next chapter.

### Indirect Action

Ionization of water causes it to lose an electron and produces a positively charged ion radical H$_2$O$^+$• and a free electron e$^-$. Through a chain of chemical reactions, H$_2$O$^+$• and e$^-$ generate highly reactive free radicals such as the hydroxyl radical (OH•) and H•
H$_2$O + radiation → H$_2$O$^+$ + e$^-$
H$_2$O$^+$ H$_2$O → H$_3$O$^+$ + OH•
These free radicals can diffuse to the target molecule leading to DNA or sugar radicals that eventually produce single and double strand breaks of the DNA helix.
RH + OH• → R• + H$_2$O
The highly reactive hydroxyl radical (OH•) and is believed to be responsible for more than 2/3 of mammalian cell damage. Radiation protection drugs typically work by scavenging free radicals. These drugs are generally less effective for high LET radiation since direct action dominates.

## References

Hall, Eric and Giaccia, Amato. Radiation Biology for the Radiologist. Lippincott Williams & Wilkins. 2006

# Radiation Sensitivity of Cells

← Interaction of Radiation with Matter · Radiation Induced Damage and Repair →

## Based on Cell Cycle

Cells propagate through division through a series of events. Radiation resistance through the cell cyle correlates with the level of sulfhydryls which are natural radiation protectors.Cells are most sensitive in the M phase and often G2. Slow cycling cells are resistant in early G1 and sensitive in late G1. Cell resistance is greatest in the last S phase because of the effective repair through homologous recombination.

## Based on Function

The Law of Bergonié and Tribondeau states cells that are dividing quickly, have less specialized functions and high metabolic rate are sensitive to radiation.
| Less Resistant | Spermatogonia |
| | Epidermal Stem Cells |
| | Osteoblasts |
| | Spermatozoa |
| More Resistant | Nerve Cells |
The difference in radiation sensitivity of tissues is based on the differences in the level of specialization and proliferation inherent in their critical cells.
| Acute Responding Tissues | Late Responding Tissues |
| Gut | Brain |
| Skin | Spinal Cord |
| Bone Marrow | Kidney |
| Mucosa | Lung |
One exception to the law are T-lymphocyte cells which are sensitive to radiation and highly differentiated.
Classification of cell radiation sensitivity has also been done based on histologic observation of cell death (Casarett's Classification) and reproductive kinetics (Michalowski's Classification).

## References

Hall, Eric and Giaccia, Amato. Radiation Biology for the Radiologists. Lippincott Williams & Wilkins, 2006.

# Radiation Induced Damage and Repair

← Radiation Sensitivity of Cells · Cell Response to Radiation Mutagenesis →

## Structure of DNA

DNA helix is an organic ladder-like molecule with two strands of phosphate molecules as the backbone and four base molecules as the crosslinks: guanine (G),adenine (A),thymine (T) and cytosine (C). The biological damage of these lesions would depend on how the cell handles this damage.

## Radiation-Induced DNA Damage

Exposure of cells to radiation leads to biological damage to the DNA molecule in the cell. This damage can take the form of damages to the base molecules or breaks in the phosphate strands. Damaged DNA matters because this can prevent genes from being correctly read or cause deletions that alter the type of protein produced.

### Base Damages

Radiation can change the structure of DNA bases by damaging, destroyed or chemically modifying them.

### Strand Breaks

The breaking of a DNA strand is assumed to occur when the absorbed energy in one sugar-phosphate volume in the DNA threshold exceeds a threshold of 17.5 eV. A single strand break (SSB) can occur on either one of the DNA strands. A double strand break (DSB) is assumed to form if two SSB's are produced on opposite strands of the DNA but within 10 base pairs. DSB's can be the result of a single particle track or two independent particle tracks.
DSB's can be quantified by pulse-field-gel-electrophoresis while SSB's can be quantified by comet assay.
DSB's are more damaging to DNA than SSB's and base damages but are also less common.

### Clustered DNA damage

Clustered DNA damage is a type of damage in which multiple DNA lesions are induced within a region of a few nm. It is produced where the density of ionization/excitation is high, whereas the isolated damage would be generated where it is low.

## Repair of DNA Damage

Radiation induces large number of lesions in DNA wich must be successfully repaired before they can have an effect. The repair of DNA decreases with increasing LET of radiation.

### For double-strand breaks

The repair pathway for double strand breaks depends on the phase of the cell's cycle.
Homologous Recombination Repair (HRR)uses a second undamaged DNA helix of similar base structure as a template for repair. This process occurs in the late-S/G2 phase of the cell cycle and is error-free.
Non-homologous end joining (NHEJ) is the rejoining of the two broken ends of the DNA by deleting some of the DNA. This process occurs in the G1 phase and is more prone to error. The possible loss of genetic code sequence in this process could be a source of oncogenic lesions.

### For Single-strand breaks

A break in one strand is readily repaired using the undamaged parallel strand of the DNA with its complementary base.

### For base damage

Base Excision Repair is the main "pathway" employed to remove radiation-induced damage to bases. The mechanics involves cleaving the damaged base and its replacement with an undamaged base through several steps including the excision of a section of the DNA sequence and its resynthesis using the complementary chain as a template.

## References

Chaudhry MA. Base excision repair of ionizing radiation-induced DNA damage in G1 and G2 cell cycle phases. Cancer Cell International 2007;7:15. doi:10.1186/1475-2867-7-15. PMCID: 2063494

# Cell Response to Radiation Mutagenesis

← Radiation Induced Damage and Repair · Mathematics of Radiation Biology →

## Cell death

Cell death of non-proliferating (static) cells is defined as the loss of a specific function, while for stem cells and other cells capable of many divisions it is defined as the loss of reproductive integrity (reproductive death).
The cell commits a pre-programmed suicide called 'apoptosis' before or after it divides or dies when attempting mitosis.

## Division delay

The cell is arrested and repaired in the late G2 phase by the p53 checkpoint gene prior to mitosis.

## Malignant Transformation

The cell survives with mutations that active a loss of the function of gene groups for tumor suppressions, as well as, DNA stability and gain in the function of gene groups for proliferation - oncogenes. These series of events could lead to the progression of cancer.

## Foetal Effects

Ionizing radiation can produce adverse pregnancy outcomes depending on the stage of gestation at the time of exposure. In utero radiation exposure is particularly harmful during the period of organogenesis during early gestation and 2nd trimester. Examples of radiation effects include intrauterine death, anencephaly, microcephaly and mental retardation.

## Genetic Effects

The mutation stays dormant then appears as hereditary effects. This usually has a time line of generations.

## Adaptation

The irradiated cell is stimulated to react and become more resistant to subsequent irradiation.

# Mathematics of Radiation Biology

← Cell Response to Radiation Mutagenesis · Imaging Tumour Physiology →

## Metrics
### Ionization Density: Linear Energy Transfer

Ionization from radiation in biological material leads to a random and uneven distribution of deposited energy in cells. The spatial distribution of the energy imparted by the charged particle is quantified by the Linear Energy Transfer (LET) metric. It is the quotient of the average energy imparted and the distance traversed by the radiation with units of keV/μm.
Radiation can be reclassified into low LET or high LET radiation based on their LET value. The demarcation value between low and high LET is about 10 keV/μm.
| Low LET Radiation | High LET Radiation |
| X ray | Alpha Particle |
| Gamma ray | Neutron |
| | Proton |

### Relative Biological Effectiveness

As the LET of radiation increases, the ability of the radiation to produce biological damage also increases. The relative biological effectiveness (RBE) compares the dose of test radiation to the dose of standard radiation to produce the same biological effect. The standard radiation has been taken as 250 kVp X rays for historical reasons, but is now recommended to be Cobalt 60 gamma rays.
Mathematically, the RBE is defined by the following ratio:
    $RBE={\frac {Dose_{s}}{Dose_{t}}}$
where $Dose_{s}$ is the dose from standard radiation to produce an effect and $Dose_{t}$ is the dose from test radiation to produce the same effect.
The RBE peaks when the separation between ionizing events coincides with the diameter of the DNA double helix (~ 2 nanometers).

### Oxygen Enhancement Ratio

In the presence of molecular oxygen (as little as a few hundred ppm) damage to DNA caused by free radicals can become "fixed" (i.e. permanent). This oxygen effect is considered since two-thirds of DNA damage is caused by free radicals.Tumor cells that are oxygen depleted (i.e. hypoxic) are thus more highly resistant to ionizing radiation.
The oxygen enhancement ratio determined by calculating the ratio of doses in hypoxic and normaxic conditions for a given isoeffect. Mathematically, it is expressed as:
    $OER={\frac {Dose_{h}}{Dose_{n}}}$
where $Dose_{h}$ is the dose to produce an effect in hypoxic conditions and $Dose_{n}$ is the dose to produce the same effect in normoxic conditions.
The oxygen enhancement ratio (OER) is typically lower for high LET radiation than for low LET radiation. The OER for electrons produced by x-rays may be as high as 3 while that for alpha particles is close to unity.

### Biologically Effective Dose
## Cell Survival Curves

The biological effects of radiation have historically been measured with cell survival curves. These curves model the relationship between a given dose of radiation and the fraction of cells surviving in cell cultures. Examples of cell-survival curves are shown on the left.
Several mathematical methods have been developed to define the shape with the Linear Quadratic Model being most used.

### Linear Quadratic Model

This model assumes there are two main ways to generate double strand breaks and subsequently cell death. The first way is caused by a single particle breaking both strands and is proportional to dose. This is the linear component of cell killing. The second way involves two independent breaks in opposite strand and is proportional to dose squared. This is the quadratic component of cell killing.
The combined effect of the linear and quadratic components of cell kill on the cell surviving fraction is given by
where alpha is a constant describing the linear component while the small constant beta describes the quadratic component. Alpha has units of Gray$^{-1}$ while beta has units of Gray$^{-2}$.
The ratio of alpha/beta gives the dose at which the linear and quadratic components of cell killing are equal. The typical values for tumours are ~3Gray and ~10Gray for normal tissues.

# Imaging Tumour Physiology

← Mathematics of Radiation Biology

## Tumor Hypoxia
### Magnetic Resonance Imaging Based

Oxygenated haemoglobin has different magnetic properties than its de-oxygenated form. These magnetic differences can be detected and imaged using a technique called Blood Oxygen Level Dependent (BOLD) Magnetic Resonance Imaging.BOLD characterization of haemoglobin oxygen saturation can be used to reflect vascular hypoxia but not tissue hypoxia$^1$.
Dynamic Contrast Enhanced (DCE-MRI) with gadolinium diethylene-triamine penta-acetic acid as the contrast agent can be used to image hypoxic fraction. MRI T1-weighted images are taken before and after an intravenous injection of gadolinium$^2$.

### Positron Emission Tomography Based

Positron Emission Tomography combined with the hypoxia PET reagent 18F misonidazole$^3$ or 60Cu-ATSM$^4$ (copper-60 labeled diacetyl-bis( N(4)-methylthiosemicarbazone))$_4$.

### Optical Based

Optical spectroscopy measures haemoglobin saturation $^5$ which is correlated to vascular hypoxia. This technique is limited to imaging sites that offer optical access.

## Interstitial fluid pressure

Dynamic Contrast Enhanced (DCE) Magnetic Resonance Imaging can be used to image the interstitial fluid pressure in tumors using gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA) as the contrast agent.

## Metabolism

* Fludeoxyglucose (18F) abbreviated to FDG can be used with Positron Emission Tomography to image tumour metabolism.
* Lactic acid is the product of anaerobic metabolism. Lactic acid metabolism can be measured using hyperpolarized $^{13}$C-pyruvate.

## References

1. Duong TQ. Cerebral blood flow and BOLD fMRI responses to hypoxia in awake and anesthetized rats. Brain research 2007;1135(1):186-194. doi:10.1016/j.brainres.2006.11.097. PMCID 2949962
2. Egeland, T. A.M., Gulliksrud, K., Gaustad, J.-V., Mathiesen, B. and Rofstad, E. K. (2012), Dynamic contrast-enhanced-MRI of tumor hypoxia. Magn Reson Med, 67: 519–530. doi: 10.1002/mrm.23014
3. Yang, David J., et al. "Development of F-18-labeled fluoroerythronitroimidazole as a PET agent for imaging tumor hypoxia." Radiology 194.3 (1995): 795-800.
4. Dehdashti, Farrokh, et al. Assessing tumor hypoxia in cervical cancer by PET with 60Cu-labeled diacetyl-bis (N4-methylthiosemicarbazone). Journal of Nuclear Medicine 49.2 (2008): 201-205.
5. Culver, Joseph P., et al. "Diffuse optical measurement of hemoglobin and cerebral blood flow in rat brain during hypercapnia, hypoxia and cardiac arrest." Oxygen Transport To Tissue XXIII. Springer US, 2003. 293-297.