Radiology I

Radiation and Cellular Effects

Ionizing Radiations
Excitation -- raising of e- to a higher energy level without ejecting it Ionization - ejection of 1 orbital eLocal release of large amounts of energy Types of radiation: -Electromagnetic -gamma raystravel at speed of light, no charge -X rays -ultra-violet -visible -infrared -radiowaves -Particulate -p+, neutrons, e-, alpha particles, beta particles Photon energies of >124eV, wavelengths of <10-6 cm

Indirect Action
For Low LET radiation, 67% damage is indirect action For High LET radiation, most (all?) damage is direct action Critical distance of indirect action is within 2nm radius from DNA.

International System Radiation Units

Radioactivity Old= Curie (Ci) New = Becquerel (Bq) 1Bq=27pCi 1Ci=3.7x1010Bq (37GBq)

Absorbed Dose

Old=rad New= Gray (Gy) 1Gy=100 rad Old=rem New=Sievert (Sv) 1Sv = 100 rem

Equivalent/Effective Dose

Definitions
Bq-2.7x10-11 Ciequal to one disintegration per sec Ci3.7x1010 disintegrations per sec Gyabsorbed radiation dose, the quantity which deposits 1 Joule of energy per kg1Gy=100rad Sievertdose equivalencemultiply absorbed dose in Gy by the Quality Factor (Q) 1Sv=100rem LETmeasure of the rate of energy transfer from an ionizing radiation to the target materialkeV of energy lost/micron track length

Dose Equivalent
Rem (Roentgen equivalent man) Biological activity of energy deposition 1 rem = 1 rad x RBE (relative biological effectiveness) RBE is usually compared with 200keV of x-rays; RBE depends of LET, dose, dose rate, biological system, etc. RBE can be replaced by Wr (radiation weighting factor) or Q or quality factor 1 seivert (Sv) = 100 rem

 Chromosome Aberrations
Cell is irradiated early in interphase before duplication of genetic material Best monitor for exposure to radiation in weeks after exposure.

Chromatid Aberrations
Cell is irradiated late in interphase, after duplication of genetic material
Radiation-induced cell killing correlates best with the induction of assymmetrical exchange aberrations. Chromosome aberrations that involve symmetrical exchanges are generally not lethal.

Lethal aberrations:

Ring dicentric

chromosome
chromosome

Anaphase bridge

Asymetric aberrations

Chromatid)

Non-lethal Chromosomal Aberrations

Symmetric Aberration Most common aberration following low doses of radiation (single hit) are terminal deletions.

Frequency of chromosomal aberrations is a linear quadratic function of dose. Low Dose: both aberrations caused by the same eHigh Dose: caused by different electrons

VIMVegetative Intermitotic Cells DIMDividing Intermitotic Cells RPMReverting Post-mitotic Cells FPMFixed Post-mitotic Cells

NOTE: Lymphocytes are considered radiosensitive!!

Medical Radiobiology Elizabeth Travis Pg 71

Clonogenic Cell Survival Assays:

Plating efficiency:
% cells seeded that grew into colonies

Surviving fraction:
Colonies counted____ (cells seeded)x(PE/100)

Comparison of Survival curves: Linear vs. Semi-Log plot

L-Q Model

Target Theory Model

Multiple doses: repair Between doses Series of single dose Survival curves

D10=2.3x D0= Dose to kill 90% of cells 2.3 is the ln10

2 1

Survival in apoptotic and non-apoptotic cells

Ladder for detection of apoptosis

Length of Time in Phases of the Cell Cycle

Cells:
Phase of cell cycle Tc CHO cells (hours) 11 HeLa cells (hours) 24

Tm
Ts TG2 TG1

1
6 3 1

1
8 4 11

Variation from one cell type to another is greatest for G1 phase.

Experiments of Warren Sinclair: Survival curves during cell cycle Shoulder vs no shoulder

----- calculated for -hypoxic conditions of -Mitotic cells

Late Sleast sens.

M>G2>G1>early S>late S for sensitivity Difference caused by cell cycle are similar to difference caused by Oxygen effect

Comparisons of CHO/HeLa Cells

Additional structure to curve here

Differences in HeLa/CHO curves are due to differences in length of time cells spend In G1 phase of the cell cycle.

General Conclusions
1. Cells are most sensitive to radiation at or close to M 2. Cells are most resistant to radiation in late S 3. For prolonged G1 a resistant period is evident early G1 followed be a sensitive period in late G1 4. Cells are usually sensitive to radiation in G2 (almost as sensitive as in M) There are exceptions to these rules in some cell lines.

Categories of Radiation Damage

1. Lethalirreversible, irreparable, leads to cell death 2. Sublethal (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 Sublethal Damage Repairincrease survival when a dose is fractionated over time 3. Potentially Lethal Damage (PLD)can be modified by the post-irradiation environment

Potentially Lethal Damage

Dose would usually be lethal, but modulation of post-irradiation environment increases survival Factors known to increase survival by affecting PLD: 1. Incubation in Hanks Balanced Salt Solution for several hours after irradiation 2. Grow cells in density inhibited state for 6-12 h following irradiation 3. Growth conditions for cells are suboptimal Relevance to therapy has been questioned

Potentially Lethal Damage in Irradiated Cells

Increase cell survival if cells Are left for 6-12h in Stationary phase

Experiment: density arrested cells Are treated with trypsin and then plated At 0, 6, 12 hours following radiation

6-12 hours

0 hours

Sublethal Damage Repair

Increased survival when a dose is split into two or more fractions separated by a time interval There is a point at which an increase in the number of fractions will no longer increase survivalplateau in the response

Plot of time between Doses in CHO cells and Survival. Note that Surviving fraction is high With >2hours between Doses.

Survival of CHO cells exposed to 2 Fractions of x-rays 3Rs: Repair Reassortment Repopulation

SLDR occurs in the first 2 hours following radiation exposure.

Dose-Rate Effect in CHO Cells

Broad shoulder to survival curve

Dose rate effect is more dramatic in CHO than in HeLa Cells

Composite survival curves for 40 Human Cell Lines

Less variation

Low Dose Rate: Survival Curves show greater variation, Greater range in repair times

Dose-rate Effects
At very low dose-rates the following occurs: Increased survival Decreased mutations Decreased chromosomal aberrations Increasing life-span Decreased pathology EXCEPT exposure of the fetus in utero where increasing the LENGTH of exposure (at low dose-rates) increases the pathology Testis is also a dose-rate responsive tissue and has more damage at low dose-rates than high dose-rates.

The Oxygen Effect Oxygen modifies the biological effects of ionizing radiation OER oxygen enhancement ratio: ratio of hypoxic: aerated doses needed to achieve the same biological effect X-Rays/-Rays at high doses is 2.5-3.0 Rapidly growing cells: OER is ~2 at lower doses

Variation of OER during cell cycle: G1 lower OER than 2

OER is absent for high LET radiations like alpha-particles and is intermediate for fast n0. 02 effect does not require that 02 be present during radiation just added 5 msec after

Cells are more sensitive to Radiation in the presence of Oxygen than in its absence

High dose region of survival curve

Low dose region of survival curve

Oxygen Concentration and Sensitivity to Radiation Approx. partial pressure of O2 at which gamma-irradiated cells exhibit radiosensitivity halfway between their fully aerobie and fully hypoxic response is near 3mmHg Cells reach full radiosensitization at 20-40mmHg, near the O2 concentration in venous blood.

1mmHg=1 Torr

Radiosensitivity increases until about 30mmHg is achieved, and then it remains the Same for all oxygen concentrations. The most dramatic increase is seen at low O2 Concentrations near 0.5% (3mmHg).

Staining of chronic hypoxic cells with nitroimidazole

Hypoxia
Selects for cells with decreased apoptotic potential Hypoxia is associated with an increased metastatic potential Hypoxic cells have an increased mutation frequency compared to oxic cells. pH in hypoxic regions is decreased (around pH6.2) compared to oxic regions Tumor-associated macrophage tend to localize to hypoxic regions.

Diffusion of Oxygen through Tumor Tissue

Oxygen can generally diffuse 70um at the arterial end of the capillary and less at the venous end.

Process of Reoxygenation

After each dose of radiation The number of hypoxic cells is Reduced, but the percentage Remains the same.

Percentage of anoxic cells In a tumor at varying times following a dose of 10Gy of X-rays.

At 6h following rad Exposure (10Gy), The remaining cells are 100% hypoxic because Radiation selectively kills Oxic cells; these cells Will redistribute in Oxygen content.

LET
Energy transferred per unit length of the track keV/m avg. energy locally imparted to medium dE

LET (L) = _________________________ =

distance traversed by charged particle

____

Most particles are not mono-energetic, so there is variation in radiation spectra 250kV X-rays: photons 250 keV can escape through tube LET is averaged Two ways to calculate LET average: 1. Track average divide track into equal lengths, calculate energy deposited in each, average it 2. Energy average divide track into equal energy increments and average the lengths of track over which these energy increments are averaged

Correct ranking of LET: 20MeV photons<50KeV x-rays<20MeV alpha<250KeV alpha

Relative Biological Effectiveness

Dosemeasure energy absorbed/unit mass of tissue High LET radiations tend to cause exponential survival curves Equal doses do not produce equal biological effects for different types of radiation 1Gy neutrons>>>>>1Gy gamma rays for biological effects RBEX-rays are used as the standard for comparison RBE of test radiation r compared to X-rays: D250/D5 where D250 and Dr are the doses of X-rays and test radiation required for equivalent biological effect To measure RBE, must choose a test system: LD50 x-rays=6Gy (250KeV x-rays) LD50 neutrons=4Gy RBE=6/4=1.5

Relationship of RBE and LET RBE increases to a maximum LET of 100KeV/um then falls

LET at which RBE peaks is similar for different mammalian cell types.

Cell survival of 0.8

Cell survival of 0.1 Cell survival of.01

Absolute value of RBE varies with what type of biological damage is monitored.

RBE is influenced by the following factors:

Radiation quality (LET) Radiation dose Number of dose fractions Dose rate Biological system/endpointhigher for tissues with SLDR than those that dont have it

Relationship of OER/RBE with LET

The point at which the RBE increases is where the OER drops most dramatically.

Radiation Weighting Factor

Radiation Weighting Factor (WR) allows for rapid comparisons of particular doses/qualities of radiation Absorbed dose X WR = Equivalent dose Gy Sv rads rems WR for low LET radiations (X-rays, -rays, e-) = 1.0 WR for high LET radiations (-particles, some n0) = 20.0 ICRP defined this If absorbed dose = .1 Gy or 10 rads WR = 20 then equivalent dose = 2Sv or 200 rems

Summary
Radiationquality, ionization, direct vs. indirect effects DNA damagechromosomal aberrations, chromatid aberrations, types of DNA lesions Cell survivaltypes of cell death, survival curves defined, apoptosis Cell cyclevariation in cell cycle, sensitivity throughout cell cycle, reassortment Cellular repairPLDR, SLDR, low dose rate effects Reoxygenationoxygen effects, hypoxia RBE and LETLET, tracks, RBE vs. LET, OER vs. LET, radiation weighting factors