SECTION III RADIATION CELLULAR BIOLOGY

"LAW OF BERGONIE & TRIBONIDEAU"

Cells are most sensitive to Radiation when:

• they are actively proliferating.

• they are undifferentiated.

Exceptions to this Law:

• lymphocyte
• Oocyte

The Table below lists categories of mammalian parenchymal cells in

order of decreasing radiosensitivity

b. Vegetative Intermitotic Cells: divide regularly undergo no

differentiation between division

c. Differentiating Intermitotic Cells: divide regularly differentiate to

some extent between divisions relatively radiosensitive

d. Reverting Postmitotic Cells: do not divide regularly, but are

capable of division under proper stimulus (example: liver tissue)
variably differentiated relatively radioresistant

e. Fixed Postmitotic Cells: do not divide highly differentiated most

radioresistant

CHARACTERISTICS AND RADIOSENSITIVITY OF CELL POPULATIONS

CELL TYPE CHARACTERISTICS EXAMPLES RADIOSENSITIVITY

Vegetative Rapidly dividing; Type A Most radiosensitive
intermitotic undifferentiated; do spermatogonia
(VIM) not differentiate Erythroblasts.
between divisions
Crypt cells of
intestines

Basal cells of
 epidermis
Differentiating Actively dividing; Intermediate Relatively
intermitotic more differentiated spermatogonia radiosensitive
(DIN) than VIMs:
differentiate between Myelocytes
divisions
Multipotential Irregularly dividing; Endothelial cells Intermediate in
connective more differentiated radiosensitivity
tissue(MCT) than VIMs or DIMs Fibroblasts
Reverting Do not normally Parenchymal Relatively
Postmitotic divide but retain cells of liver. radioresistant
(RPM) capability of division:
differentiated Lymphocytes*
Fixed post Do not divide; Some nerve cells Most radioresistant
mitotic (FPM) differentiated
Muscle cells

Erythrocytes
(RBCs)
Spermatozoa

*Lymphocytes, although classified as relatively radioresistant by their characteristics are very

radiosensitive.
1. GENERAL DEFINITIONS

Radiosensitivity:

The relationship between sensitivity to radiation at the

cellular level and dose.

Radioresponsiveness:

The rate a tumor shrinks after irradiation. Not a function of

radio-sensitivity but more related to rate of cell removal. i.e.
Cell Loss Factor

THE MINIMAL TOLERANCE DOSE:

TD 5/5: Dose which will produce 5% complication in 5

years.

THE MAXIMAL TOLERANCE DOSE:

TD 50/5: Dose which will produce 50% complications in 5

years.

Mechanisms of Radiation Damage—In an organ damage occurs in

both parenchymal and stromal cells and radiosensitivity of the
organ is defined by the most radiosensitive cells or tissues within
that organ.

Example: Radiosensitive Organ (testis)

 -Damage to parenchymal cell is indirect via vascular compromise.

EVALUATING RADIOSENSITIVITY
Various organs in decreasing order of radiosensitivity based on
hypoplasia in the parenchyma of the organ two months postirradiation*

ORGAN RADIOSENSITIVITY CHIEF MECHANISM OF

HYPOPLASIA
Lymphoid organs, High Destruction of radiosensitive
bone marrow; parenchymal cells
blood, testes,
ovaries, intestines
Skin and other Fairly high Destruction of radiosensitive
organs with parenchymal cells of the epithelial
epithelial cell lining
lining (cornea, oral
cavity, esophagus,
rectum, bladder,
vagina, uterine
cervix, ureters)
Optic lens, Medium Destruction of mitotically active
stomach epithelial cell,

Growing cartilage Destruction of mitotically active

chondroblasts, plus some damage
to the fine vasculature and
connective tissues

Damage to the endothelium

Fine vasculature
Destruction of mitotically active
Growing bone chondroblasts or osteoblasts. plus
some damage to the fine
vasculature
Mature cartilage orFairly low Hypoplasia of parenchymal cells is
bones: Salivary secondary to damage to the
glands, respiratory vasculature and connective tissue
(minor contribution to hypoplasia
organs, kidneys, from direct effects on parenchymal
liver, pancreas, cells)
thyroid, adrenal
and pituitary
glands
Muscle, brain, Low Hypoplasia of parenchymal-cells is
spinal cord secondary to damage to the
vasculature and connective tissue
(minor contribution to hypoplasia
 from direct effects on parenchymal
cells)

From Rubin, P. and Casarett. G. W.: Clinical Radiation pathology

(Philadelphia: W. B. Saunders. 1968).

o Radiosensitivity depends on the "endpoint" measured

 chromosomal aberrations
 mitotic delay
 cell death
 loss of chemotactic ability
 degranulation

o Conditional vs inherent sensitivity

 cell cycle
 oxygenation

2. Target Theory. General Concepts.

o Calls contain critical Biological targets

o DNA: most important (critical target molecule)
o Destruction (inactivation) of more than one critical target
may be necessary to produce call death (loss of reproductive
integrity) called "multitarget system"
o More than one "HIT" (Ionization event in the critical target)
may be necessary to inactivate a target. Called "multihit
system"

3. Single Target - Single Hit Model.

o One "HIT" will cause cell death

o Radiation interactions are random thus the probability of a
"HIT" follows a poison statistics
o As radiation impinges on a group of cells some cells will
experience radiation interactions outside the critical target
(DNA). This is the so-called "wasted dose."
4. ORGAN SYSTEM RESPONSE

b. Definitions
 Dose Severity
 Low: 0 - 1 Gy (0 - 100 rads)
 Moderate: 1 – 10 Gy (100 - 1,000 rads)
 High: > 10 Gy (1,000 rads)
 Response of Tissues to Radiation
 Morphological changes
 Functional changes
 No "unique" radiation specific response
 Expression of damage is, time dependent
c. Schematic of Biological Response to Radiations
 Replacement of damaged cells by replacement of damaged
different cell type (i.e., fibrosis) cells by same cell type
organ is not returned to its pre- organ returned to pre-
irradiated state irradiated state
radioresistant tissues (FPM cell types) radiosensitive tissue (VIM,
can only affect repair, e.g. muscle & CNS DIM, multipotential, e.g. skin,
SI, bone marrow)
*Note: If doses are high, germinal layer is destroyed, therefore, regeneration ability is
lost + repair (fibrosis)

o Time vs. Response

 Radiosensitive Tissue -----> Early Response
 Initial Response < 6 months are the results of
cell damage via
a. mitotic delay
b. reproductive failure
c. interphase death
 Changes are occurring in both the parenchymal
(functional) and stromal (supportive); however,
early expression is typically loss of parenchymal
integrity
 Radioresistant Tissues -----> Late Response Result of
progressive and irreversible damage to stromal tissue
which eventually lead to parenchymal death.
 Example: Skin & Lung Irradiations 20 Gy (2,000 rads)

Skin: (sensitive) --- > (erythema) ---> (sloughing) --- >

(normal)

Lung: (resistant) --- > (no significant change) --->

(progressive lung fibrosis)

Note: Progression of vascular damage in resistant organs is

slow

Therefore, response is a function of healing (repair and

regeneration) which is dependent on the cells.

 If the D37 (D0) dose is delivered twice to a group of cells

14% (.37 x .37) of the cells would be expected to
survive etc.

 Thus for a particular cell line

 A low D37 represents a "radiosensitive" cell line.
 A high D37 represents a "radioresistant" cell line.

 D37 for mammalian cells in culture and low L.E.T.

radiation= 1 Gy (100 rads)
 D37 high L.E.T. = 100 mGy (10 rads)

 Mathematical expression of surviving cell population

(S) after a given dose or radiation is identical to the
radioactive decay law (i.e., a first order exponential
relationship)

S = e or In S = KD
KD

6. Multi-target - Single Hit Model

a.A "Target" can be "hit" at least once without loss of cell

survival.

b.This represents a "Quasi Threshold Dose" (DQ) below which

the effect seen is diminished
c. Typically thought to represent complex biological systems.
Capable of repair.

d. The dose (DQ) produces "sublethal" damage

e. There are often several different structures which may

control the same function. Therefore, the inactivation of the
function by a single hit is highly unlikely.

f. This is not to say that one hit is not sufficient to inactivate

anyone of the targets, but that sufficient active targets will
remain to carry on the function, thus the system as a whole
appears unaffected.
 g. Extrapolation Number (n) = # of targets/cell or # of
hits/target to inactivate the cell. For mammalian cells in
culture n= 2-10.

7. Other Direct Cellular Responses

a. Radiation effects on cell membranes

Large doses 30-50 Gy (3-5 K rads) may cause plasma

membranes to rupture and membranous structures of the
endoplasmic reticulum to dilate. The membrane surrounding
the lysosome may disintegrate thus releasing the lysosome
contents and killing the cell.

 At lower doses subtle changes in the transfer of K+,

Ca2+ and Na+ ions may occur causing an electrolytic
imbalance resulting in function impairment or even
death.

b. Enzyme inactivation

 In general, high doses of radiation are required before

significant enzyme activation takes place.

 Inactivation of enzymes does not appear to be a major

mechanism for cell death, at least in the low to
moderate dose range.

c. Other cell constituents

 Carbohydrates and lipids in the cells are not very

radiosensitive.

 Proteins tend to be more radiosensitive than

carbohydrates and lipids.

7. Effect of LET, RBE and OER

a. At high LET: Cell survival kinetics follows the single target-
single-hit model.
b. RBE α LET
 Oxygen promotes damage: RO° (free radicals) and thus
↓ D0
  Possible Explanations
 S phase has highest levels of endo and
exonuclease (i.e. repair capability)
 S & associated with concentration of free
radical scavengers, e.g. sulfhydryl compounds
like cysteine: SH - CH2 – CH
 DNA tightly coiled in M, therefore probability for
lethal hit.

FACTORS AFFECTING RESPONSE

PHYSICAL CHEMICAL BIOLOCIC

LET Sensitizers Cell Cycle
Dose-rate Oxygen Intracellular repair
Halogenated pyrimidines
Others-partial sensitizers
Protectants
Cysteine
Cysteamine
Figure 3-18. Summary of survival curves for clonogenic assays of cells from normal
tissues. The human ataxia-telangiectasia cells are included because they are the most
sensitive mammalian cells. The bone marrow colony-forming units, together with the
mammary and thyroid cells, represent systems in which cells are irradiated and assayed
by transplantation into a different tissue in recipient animals. The jejunal crypt and testis
stem cells are examples of systems in which cells are assayed for regrowth in situ after
irradiation.

SUMMARY OF CELL SURVIVAL CURVES

• The cell cycle for mammalian cells can be divided into four phases: mitosis (M),
followed by G1, followed by the DNA synthetic phase (S), then G2, and into
mitosis again.

• The fastest cycling mammalian cells in culture, and crypt cells in the intestinal
epithelium, have a cycle time as short as 9 to 10 hours. Stem cells in resting
mouse skin may have a cycle time of more than 200 hours. Most of this difference
is due to the varying length of G1, the most variable phase of the cycle. M and S
do not vary much.

• In general, cells are most radiosensitive in late M and G2 phases and most resistant
in late S.

• For cells with a longer cell cycle time and a significantly long G1 phase, there is a
second peak of resistance late in G1

• The pattern of resistance and sensitivity correlates with the level of sulfhydryl
compounds in the cell. Sulfhydryls are natural radioprotectors and tend to be at
their highest levels in S and at their lowest near mitosis.

• The presence or absence of molecular oxygen dramatically influences the

biological effect of x-rays.
• The oxygen enhancement ratio (OER) is the ratio of doses without and with
oxygen to produce the same biological effect.

• The OER for x-rays is about 3 at high doses and is possibly lower (about 2) at
doses of 50 to 200 rads (0.5 to 2 Gy).

• OER decreases as LET increases. The OER approaches unity (i.e., no oxygen
effect) at an LET of about 160 keV/µ m. For neutrons the OER has an
intermediate value of about 1.6.

• To produce its effect. Oxygen must be present during the radiation exposure or at
least during the lifetime of the free radicals (10-5 sec).

• Oxygen "fixes" (i.e., makes permanent) the damage produced by free radicals. In
the absence of oxygen damage produced by the indirect action may be repaired.
Oxygen modifies the indirect but not the direct action of radiation.

• Only a small quantity of oxygen is required for radiosensitization: 0.5% oxygen

results in a sensitivity halfway between hypoxia and full oxygenation.

• Tumors contain hypoxic cells because they outgrow their blood supply. By 150 to
200 µ m from a capillary the oxygen concentration is too low for cell viability,
hence the presence of necrotic areas. At the end of the oxygen diffusion range
there may be cells at an oxygen level low enough to be intransigent to killing by
x-rays but high enough to be viable.
• Most transplantable tumors in animals have been shown to contain hypoxic cells
that limit curability by single doses of x-rays. Hypoxic fractions vary from 0% to
50% but are frequently about 10% to 15%. Studies of hypoxic cell sensitizers
indicate similar proportions in human tumor nodules.

• Reoxygenation is the process by which cells that are hypoxic at the time of
irradiation become oxygenated afterwards.

• The extent of reoxygenation and the rapidity with which it occurs varies widely
for different experimental animal tumors.

• If reoxygenation is rapid and complete, hypoxic cells have little influence on the
outcome of a fractionated radiation schedule.

• Reoxygenation cannot be measured in human tumors, but presumably it occurs, at

least in those tumors controlled by conventional fractionated radiotherapy.

• The quality factor (Q) depends on LET and is specified by the International
Commission on Radiological Protection as a representative RBE at low dose and
low dose rate and relevant to late effects. It is used in radiological protection to
reduce radiations of different biological effectiveness to a common scale.

• Dose equivalent is the product of absorbed dose and quality factor. If absorbed
dose is expressed in gray (Gy), dose equivalent is in sievert (Sv). If absorbed dose
is in rads. Dose equivalent is in rems.

GENERAL SUMMARY

• X- and γ -rays are called sparsely ionizing because along the tracks of the
electrons set in motion, primary ionizing events are well separated in space.

• Alpha-particles and neutrons are densely ionizing because the tracks consist of
dense columns of ionization.

• Linear energy transfer (LET) is the energy transferred per unit length of track.
Typical values are 0.3 keV/µ m for 60Co γ -rays, 2 keV/µ m for 250 kV x-rays, and
100 to 2000 keV/µ m for heavy charged particles.

• Relative biological effectiveness (RBE) of some test radiation (r) is the ratio
Dx/Dr where Dx and Dr are the doses of 250-kV x-rays and the test radiation
respectively, required to produce equal biological effect.

• RBE increases with LET to a maximum at about 100 keV/µ m, thereafter

decreasing with higher LET because of "overkill."
 • The RBE of high-LET radiations compared with low-LET radiations increases as
the dose per fraction decreases. This is a direct consequence of the fact that the
dose response curve for low-LET radiations has a broader shoulder than for high-
LET radiations.

• RBE varies according to the tissue or endpoint studied. In general RBE values are
high for cells or tissues that accumulate and repair a great deal of sublethal
damage, so that their dose-response curves for x-rays have a broad initial
shoulder.

• RBE depends on:

o Radiation quality (LET)
o Radiation dose
o Number of fractions
o Dose rate
o Biological system or end point

From Hall, 1994.