Radiobiology for the Radiologist, chapter 6, pg 91 - 111
The Oxygen Effect and
Reoxygenation
1
The Nature of the Oxygen Effect
2
The Nature of the Oxygen Effect

Survival curves for mammalian cells exposed to xrays in the presence and absence of oxygen

Sensitivity to x-rays



Aerated → S↑
Hypoxia → S↓
Oxygen enhancement ratio
(OER)


The ratio of hypoxic to
aerated doses needed to
achieve the same biological
effect
High dose (dose > 2Gy)

OER = 2.5 – 3
3
The Nature of the Oxygen Effect

Low dose (dose < 2 Gy)


OER = 2
Reasons:




Variation of OER with the
phase of the cell cycle
OER (G1 phase) < OER (S
phase)
G1 more radiosensitive
Dominate the survival at low
dose region
4
The Nature of the Oxygen Effect

The oxygen enhancement ratio (OER) for various
types of radiation
Large & important
Intermediate
Absent
5
The Time at which Oxygen Acts
6
The Time at which Oxygen Acts

Experiment for the time at O2 acts




O2 at high pressure chamber
“explode” onto single layer bacteria
At various time before or after irradiation
Result:


Oxygen need not be present during the irradiation
to sensitize
Could be added afterward
7
Mechanism of the Oxygen Effect
8
Mechanism of the Oxygen Effect

Chain of events from the absorption of radiation
Absorption of radiation
Production of fast charged particles
Passing through biologic material
Production of a number of ion pairs (10–10 sec)
Free radicals (10–5 sec)
Break chemical bond
Biological damage
9
Mechanism of the Oxygen Effect

O2 acts at the level of the free radicals

Reacts with the free radical



R‧+ O2 → RO2‧
Form organic peroxide
Oxygen fixation hypothesis


The damage produced by free radicals in DNA can be
repaired under hypoxia
May be “fixed” if molecular oxygen is available
10
The Concentration of Oxygen Required
11
The Concentration of Oxygen Required


Survival curve for Chinese hamster cells
exposed to x-rays in
the presence of various
oxygen concentrations
Result


100 ppm,
noticeable in change
2200 ppm, halfway
toward the fully
aerated condition
○
Air
●
2200 ppm, 1.7 mmHg
□
355 ppm, 0.25 mmHg
■
100 ppm, 0.075 mmHg
△
10 ppm, 0.0076 mmHg
12
The Concentration of Oxygen Required

The dependence of radiosensitivity on oxygen
concentration

Most of this change
of sensitivity


Further increase


Increase from
0 – 30 mmHg
little further effect
Sensitivity halfway

pO2 : 3mm Hg
13
The Concentration of Oxygen Required

Conclusion


Very small amounts of oxygen are necessary to
produce the dramatic and important oxygen effect
observed with x-rays
Oxygen tension of the body tissues



Venous blood or lymph → 20 – 40 mmHg
Different tissues may vary over a wide range from
1 – 100 mmHg
Borderline hypoxic tissue, e.g. liver, skeletal
muscle
14
Chronic Hypoxia
15
Chronic Hypoxia

Definition


Result from the limited diffusion distance of
oxygen through tissue that is respiring
First described by Thromlison and Gray


Specimen : bronchial carcinoma
Cell of the stratified squamous cell
carcinoma
16
Transverse section of tumor cord.
Stroma
Surrounded by
intact tumor cells
Central necrosis
A typical tumor area in which necrosis is not far advanced.
17
Bands of tumor cells
Stroma
Large areas of necrosis
Large areas of necrosis separated from the stroma by bands
of tumor cells about 100 mm wide
18
Chronic Hypoxia

The conclusion


Small tumor cord

Radius < 160 μm

No necrosis
Tumor cord > 200 μm


Present of necrotic center
Tumor cord enlarged further

Thickness of the sheath of viable tumor cells remained
essentially constant (100 – 180 μm)
19
The diffusion of oxygen from a capillary through tumor tissue
★
★
O2 is high enough for the
cells to be viable
O2 is low enough for them
to be relative protected
from the effects of x-rays
These cells may limit the
radiocurability of the
tumor
Proposed solution
High pressure oxygen
chamber
Neutrons
Negative π-mesons
Heavy charged ions
20
Acute Hypoxia
21
Acute Hypoxia

Definition




Develop in tumors as a result of the temporary
closing or blockage of a particular blood vessel
Tumor blood vessels open and close in a random
fashion
Different regions of the tumor become hypoxic
intermittently
First postulated by Brown in 1980s
22
Acute
hypoxia
Chronic Hypoxia
Acute Hypoxia
• Result from
temporary
closing of tumor
blood vessels
• The cells are
intermittently
hypoxic
• Normoxia is
restored each
time the blood
vessel opens up
again
Diagram illustrating the difference between chronic and acute hypoxia
23
The First Experimental Demonstration of
Hypoxic cells in a tumor
24
The First Experimental Demonstration
of Hypoxic cells in a tumor




By Powers and Tolmach
Technique: dilution assay technique
Aim: investigate the radiation response of a
solid subcutaneous lymphosarcoma in the
mouse
Survival estimates: between 2 – 20 Gy
25
First component
Dose : < 9 Gy
Slope (D0) : 1.1 Gy
Second component
Dose > 9 Gy
Slope (D0) : 2.6 Gy
2.5 time shallower
Fraction of surviving cells as a function of dose for a solid
subcutaneous lymphosarcoma in the mouse irradiated in vivo
26
The First Experimental Demonstration
of Hypoxic cells in a tumor

The survival curve consists of two separate
component

Strongly suggests that the tumor consist of two
separate groups of cells



Oxygenated cells
Hypoxic cells
The shallow component of the curve cut the
surviving fraction axis


Survival level : 1%
Means: 1% of the clonogenic cells in the tumor
were deficient in O2
27
The First Experimental Demonstration
of Hypoxic cells in a tumor

At lower doses


At higher doses



dominated by the killing of the well-oxygenated
cells
Oxygenated cells are depopulated severely
The response of the tumor is characteristic of the
response of hypoxic cells
Conclusion


A solid tumor could contain cells sufficiently
hypoxic to be protected from cell killing by x-rays
Still clonogenic and capable for tumor regrowth
28
Proportion of Hypoxic cells in Various
Animal Tumors
29
Proportion of Hypoxic cells in Various
Animal Tumors

Moulder and Rockwell




Published a survey of all published data in
hypoxic fractions
42 tumor types studies
37 tumor types contain hypoxic cells
Hypoxic fraction:



range from 0 – 50%
Average: about 15%
Dische and Denekamp


Proportion of hypoxic cells in human
Consistence with the 10 – 15 % characteristic of
many animal tumors
31
Evidence for Hypoxia in Human
Tumors





Analogy can be made with mouse tumors, in which
hypoxia can be demonstrated unequivocally.
Histologic appearance suggests the possibility of
hypoxia
Blinding of radioactive-labeled nitroimidazoles occurs
Oxygen-probe measurements are predictive
Pretreatment hemoglobin levels are powerful
prognostic factor in SCC of the cervix, carcinoma of
the bronchus, and TCC of the bladder
32
Reoxygenation
33
Reoxygenation

Van Putten and Kallman


Determined the proportion of hypoxic cell in
mouse sarcoma without irradiation and after
various fractionated radiation treatment.
Result:

Proportion of hypoxic cells




Untreated : 14%
1.9 Gy/Fx/Day × 5 days : 18% (test in 3 days later)
1.9 Gy/ Fx/Day × 4 days : 14% (test in 1 day later)
The proportion of hypoxic cells is about the
same.
34

Reasons:




A dose of x-rays kills a
greater proportion of
aerated than hypoxic cells
More radiosensitive
After oxygenation,
preirradiation pattern tends
to return
Significant of fractionation


Allow sufficient time for
oxygenation
The presence of hypoxic
cells does not greatly
influence the response of
tumor
35
Time Sequence of Reoxygenation
36
Time Sequence of Reoxygenation

Immediately after
irradiation
100% of viable
cells are hypoxic
Percentage of hypoxic
cells in a
transplantable mouse
sarcoma as a function
of time after a dose of
10 Gy of x-rays

By Kallman & Bleehen
By 6 hours, percentage of
hypoxic cells has fallen to a
close value to the
preirradiation level
37
△
Mouse osteosarcoma
●
▲
mouse fibrosarcoma
Rat sarcoma
○
Mouse mammary carcinoma
The extent and rapidity
of reoxygenation is
extremely variable and
impossible to predict
Cell type
Reoxygenation
Mammary
carcinoma
Rapid & well
Rat sarcoma
Two waves
Osteosarcom
a
Not all cells &
slow
Fibrosarcom
a
Rapid but not
complete
The proportion of hypoxic cells as a function of time after irradiation
with a large dose for 5 transplanted tumors in experimental animals
38
Mechanism of Reoxygenation

In chronic hypoxia






Cell killed by radiation are broken down and
removed from tumor population
Restructuring or a revascularization of tumor
Tumor shrinks in size
Closer to blood supply
Taking place over period of days as the tumor
shrinks
In acute hypoxia


Blood vessel is temporarily closed during
irradiation
Quickly reoxygenate when that vessel reopens
39
The Important of Reoxygenation in
Radiotherapy
40
The Important of Reoxygenation in
Radiotherapy

The reoxygenation studies with C3H mouse
mammary carcinoma



Reoxygenation 2 – 3 days after irradiation
The proportion of hypoxic cells
is lower than in untreated tumors
Prediction

Several large dose of x-rays
given at 48 hours intervals
would virtually eliminate the
problem of hypoxic cells in
this tumor
41
The Important of Reoxygenation in
Radiotherapy

Fowler and his colleagues



The x-ray schedule for cure of this tumor
Five large doses in 9 days
Suggestion


X-irradiation can be an extremely effective form of
therapy
But ideally required optimal choice of fractionation
pattern
42
The Important of Reoxygenation in
Radiotherapy

Demands a detailed knowledge of the time
course of reoxygenation in the particular tumor
to be irradiated



Evidence from radiotherapy clinic



Available for only a few animal tumors
Impossible to obtain for human
Eradication doses for many tumors
60 Gy in 30 fractions
Hypothesis


Human tumors do not respond to conventional R/T
Do not reoxygenate quickly and efficiently
43
Hypoxia and Tumor Progression
44
Hypoxia and Tumor Progression

Clinical study in Germany



Correlation between local control in advance
carcinoma of the cervix, treated by R/T
Using O2 probe measurement
Result



pO2s > 10 mmHg → local control↑
pO2s < 10 mmHg → local control↓
Suggestion

Hypoxia is a general indicator of tumor aggression
45
Hypoxia and Tumor Progression

Another study in United State



Soft-tissue sarcoma for R/T
Correlation between tumor oxygenation and the
frequency of distant metastases
Result



pO2s > 10 mmHg → distant metastasis 35 %
pO2s < 10 mmHg → distant metastasis 70 %
Conclusion

Level of tumor oxygenation influences the
aggressiveness of the tumor
46
Hypoxia and Tumor Progression
Inactivation → p53 tumor suppression gene
Overexpression → bcl-2 antiapoptotic gene
Illustration how hypoxia is linked with malignant progression
47
The End
Thanks for your attention !
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