Radiobiology for the Radiologist, chapter 6, pg 91 - 111

The Oxygen Effect and

Reoxygenation

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The Nature of the Oxygen Effect

2
 The Nature of the Oxygen Effect
 Survival curves for mammalian cells exposed to x-rays 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
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 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

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The Concentration of Oxygen Required

11
 The Concentration of Oxygen Required
val curve for Chinese hamster cells
ed to x-rays in
resence of various
n concentrations
t
0 ppm,
ticeable in change
00 ppm, halfway
ward the fully
rated condition

○ Air
● 2200 ppm, 1.7 mmHg
□ 355 ppm, 0.25 mmHg
■ 100 ppm, 0.075 mmHg
△ 10 ppm, 0.0076 mmHg

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 The Concentration of Oxygen Required
 The dependence of radiosensitivity on oxygen concentration
 Most of this change
of sensitivity
 Increase from
0 – 30 mmHg
 Further increase
 little further effect
 Sensitivity halfway
 pO2 : 3mm Hg

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 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

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Chronic Hypoxia

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 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

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 Transverse section of tumor cord.

Stroma

Surrounded by
intact tumor cells
Central necrosis

A typical tumor area in which necrosis is not far advanced.

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 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
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 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)

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 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

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 Acute Hypoxia
Acute
hypoxia • Result from
Chronic Hypoxia
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

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The First Experimental Demonstration of
Hypoxic cells in a tumor

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 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

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 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
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 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
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 The First Experimental Demonstration
of Hypoxic cells in a tumor
 At lower doses
 dominated by the killing of the well-oxygenated cells
 At higher doses
 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

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Proportion of Hypoxic cells in Various
Animal Tumors

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 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

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 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

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Reoxygenation

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 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.
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 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

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Time Sequence of Reoxygenation

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Time Sequence of Reoxygenation
 Percentage of hypoxic cells in a
transplantable mouse sarcoma as a
function of time after a dose of 10 Gy
Immediately after of x-rays
irradiation  By Kallman & Bleehen
100% of viable
cells are hypoxic

By 6 hours, percentage of
hypoxic cells has fallen to a
close value to the
preirradiation level
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 △ Mouse osteosarcoma The extent and rapidity
of reoxygenation is
● mouse fibrosarcoma
extremely variable and
impossible to predict

Cell type Reoxygenation

▲ Rat sarcoma Mammary Rapid & well
carcinoma
Rat sarcoma Two waves
Osteosarcom Not all cells &
a slow
○
Mouse mammary carcinoma
Fibrosarcom Rapid but not
a complete

The proportion of hypoxic cells as a function of time after irradiation

with a large dose for 5 transplanted tumors in experimental animals
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 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

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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
 Available for only a few animal tumors
 Impossible to obtain for human
 Evidence from radiotherapy clinic
 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

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Hypoxia and Tumor Progression

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 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

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 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

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 Hypoxia and Tumor Progression
Inactivation → p53 tumor suppression gene
Overexpression → bcl-2 antiapoptotic gene

Illustration how hypoxia is linked with malignant progression

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The End

Thanks for your attention !

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