RADIOBIOLOGY OXYGEN EFFECT & REOXYGENATION HYPOXIA IN TUMORS AND METHODS TO OVERCOME IT

1. Oxygen Effect and
Reoxygenation
Presented by:Dr. Isha Jaiswal
Moderator: Prof. M.L.B Bhatt
Date:01 September 2014

2. Overview
• Mechanism of oxygen effect
• Oxygen enhancement ratio & factors affecting OER
• Chronic and Acute Hypoxia
• Reoxygenation
• Hypoxia and Chemoresistance
• Hypoxia and Tumor Progression
• Hypoxia in clinical practice

3. Among the various clinical & pharmacological agents
that modify the biologic effect of ionizing radiation
none is simpler than oxygen,
none produces such a dramatic effect &
none has such obvious practical implications.

4. Mechanisms of oxygen effect

5. Indirect action of radiation
- 5
e-
X ray
 ray P+
O
H
H
OH-H+
Ho
OHo

6. Oxygen Acts At The Levels Of The Free Radicals.
incident X ray photon
Fast e-
Ion radical
Free
radical
Chemical changes
Biological
changes
10-15
s
10-10
s
10-9 s
10-5
s
Hours, days,
months, yrs,
generations
The chain of events:
Absorption of radiation
Production of fast charged particles
Production of ion pairs
Production of free radicals
Breakage of chemical bonds,
chemical changes,
initiation of the chain of events that
result in biological damage

7. The Oxygen fixation hypothesis.
Oxygen “fixes” (i.e. makes permanent) the damage
produced by free radicals.
The formation of RO2,an organic peroxide,
represents a non restorable form of the target material;
ie.the reaction results in a change in the chemical
composition of the material exposed to the radiation.

8. Oxygen Enhancement Ratio (OER)
The ratio of HYPOXIC to AEROBIC doses
needed to achieve the SAME biological
effects
D0 D0
0 10 20 30
Dose (Gy)
Surviving Fraction
1
0.1
0.01
Aerobic
Hypoxic
1/e
1/e
OER =
D0 (hypoxic)
D0 (aerobic)
= 3.22
OER =
D0 (hypoxic)
D0 (aerobic)

9. Parameters Affecting OER
1. Nature of cellular sensitivity
2. Nature of radiation (x-ray, neutron, alpha particle,)
3. Linear energy transfer
4. pO2
5. Cell Cycle
4. Time of oxygen presence

10. 1. Cellular Sensitivity
Radiation sensitive cells can be killed at
lower dose.
Thus, the OER is smaller.
Sensitive Cells : Low OER
The more resistant cells require large
amounts of damage.
Therefore, OER is more pronounced at
higher dose.
Resistant Cells : High OER

11. 2. Nature of radiation
 The OER for alpha particles is unity.
 X-rays exhibit a larger OER of 2.5.
 Neutrons are between with an OER of 1.6.

12. 3. OER as a function of LET

13. 4. Oxygen effect is time-dependent
 For the oxygen effect to be observed, oxygen must be present during the radiation
exposure or, to be precise, during or within microseconds after the radiation exposure.
 Oxygen sensitization occurred with oxygen added as late as 5 milliseconds after
 Oxygen acts at the level of the free radicals.
 The free radicals break chemical bonds.
Radiation Ion pairs Free Radicals
T = 10-10 sec T = 10-5 sec (0.01 msec)
irradiation.
 The extent of the damage depends on the presence or absence of oxygen.
Time delay is determined by lifetime of free radicals

14. 5. Effect of phases of cell cycle
 For rapidly growing cells cultured in vitro, the OER has a smaller value of
about 2.5 at lower doses
 Cells in G1 have a lower OER than those in S.
 Because G1 cells are more radiosensitive they dominate the low-dose
region of the survival curve.
 For this reason the OER of an asynchronous population is slightly smaller at
low doses than at high doses

15. 6. O2 concentration
 Very small amounts of oxygen are necessary to produce the dramatic and
important oxygen effect observed with X-rays.
 Oxygen tension between different tissues may vary over a wide range fro
m 1 to 100 mm Hg.
 Many tissues are bordering hypoxic and contain a small proportion of ce
lls that are radiobiologically hypoxic.
 This is particularly true of, for example, the liver and skeletal muscles

16. • By the time a concentration of oxygen
corresponding to 2% has been reached, the
survival curve is virtually indistinguishable
from that obtained under conditions of
normal aeration.
• Increasing the amount of oxygen present
from that characteristic of air to 100%
oxygen does not further affect the slope of
the curve.
3 times
Rapid change of radio sensitivity occurs from
zero to about 30 mmHg(5% oxygen)

17. Types of hypoxia

18. Chronic and Acute Hypoxia
• Hypoxia in tumors can result from two quite different
mechanisms.
• Chronic hypoxia : results from the limited diffusion distance of oxygen
through tissue that is respiring.
• Acute hypoxia : the result of the temporary closing of a tumor blood
vessel owing to the malformed vasculature of the tumor.

19. • As the tumor cord grows larger, the
necrotic center also enlarges, so that the
thickness of the sheath of viable tumor
cells remains essentially constant.
• The oxygen can diffuse in respiring
tissue is about 70 um.

20. The diffusion of oxygen from a capillary through tumor
The distance to which oxygen can diffuse is about 70 μm at the arterial end of a capillary and
less at the venous end..
The distance to which oxygen can diffuse is limited by the rapid rate at which it is metabolized by
respiring tumor cells.
Hypoxic tumor cells, lie in between (gray).
In this region, the oxygen concentration is high
enough for the cells to be viable but low enough for
them to be relatively protected from the effects of
x-rays.
These cells may limit the radiocurability of
tumor.

21. Acute hypoxia
• Regions of acute hypoxia develop in tumors as
a result of the temporary closing or blockage of
a particular blood vessel.
• Results from transient fluctuations in blood
flow because of the malformed vasculature.
• The cells are intermittently hypoxic because
normoxia is restored each time the blood
vessel opens up again

22. The First Experimental Demonstration of hypoxia in tumor
Powers and Tolmach investigated radiation response o
f a solid subcutaneous lymphosarcoma in the mouse
The survival curve for this solid tumor clearly consist
s of two separate components.
- first, up to a dose of about 9 Gy has a D0 of 1.1 Gy
-second has a shallower D0 of 2.6 Gy
This biphasic survival curve has final slope about 2.5
times shallower than the initial portion, strongly sugge
sts that the tumor consists of two groups of cells,
oxygenated and hypoxic.
If the shallow component of the curve is extrapolated
backward , it does so at a survival level of about 1%.
From this it may be inferred that about 1% of cells in
the tumor were hypoxic
D0 of 1.1 Gy
D0 of 2.6 Gy

23. • In this experiment survival estimates were made from
2 -25 gy
• The response of this tumor to single doses of radiation of
various sizes is explained readily on this basis.
• If 99% of the cells are well oxygenated and 1% are
hypoxic, the response to lower doses is dominated by the
killing of the well-oxygenated cells.
• For these doses, the hypoxic cells are depopulated to a
negligibly small extent.
• Once a dose of about 9 Gy is exceeded, the oxygenated
compartment of the tumor is depopulated and response of
the tumor is characteristic of the response of hypoxic cells.
• This biphasic survival curve was the first unequivocal
demonstration that a solid tumor could contain cells
sufficiently hypoxic to be protected from cell killing by x-rays
but still clonogenic and capable of providing a focus
for tumor regrowth.

24. Reoxygenation
• Van Putten and Kallman (experimented in mouse sarcoma)
• The proportion of hypoxic cells in the untreated tumor was about 14%.
• The ratio did not change after giving fractionated radiation.
• During the course of the treatment, some hypoxic cells become
oxygenated.
• If this was not there then the proportion of hypoxic cells should
increase
• This phenomenon, by which hypoxic cells become oxygenated
after a dose of radiation, is termed reoxygenation.

25. Process of Reoxygenaion
 Tumors contain a mixture of aerated and hypoxic cells
 A dose of x-rays kills a greater proportion of
aerated cells than hypoxic cells
 Therefore, immediately after irradiation, most cells in
the tumor are hypoxic.
 However, the preirradiation pattern tends to return
because of reoxygenation.
 If the radiation is given in a series of fractions
separaed in time sufficient for reoxygenation to occur
, the presence of hypoxic cells does not greatly
influence the response of the tumor.

26. Mechanism of Reoxygenation
• In experimental animals, some tumors take several days to reoxygenate;
in others, the process appears to be complete within 1 hour or so
• The differences of timescale reflect the different types of hypoxia that are
being reversed, chronic versus acute.
• Chronic : As the tumor shrinks in size, surviving cells that previously
were beyond the range of oxygen diffusion are closer to a blood supply
and so reoxygenate.
• Acute (complete within hours): the reoxygenation of acutely hypoxic
cells; those cells that were hypoxic at the time of irradiation because they
were in regions in which a blood vessel was temporarily closed quickly
reoxygenate when that vessel is reopened.

27. Hypoxia and Chemoresistance
• Hypoxia can also decrease the efficacy of some chemotherapeutic agents owing to
fluctuating blood flow, drug diffusion distance, and decreased proliferation.
• Some chemotherapeutic agents that induce DNA damage, such as doxorubicin
and bleomycin, are less efficient at killing hypoxic tumor cells in part because of
decreased free-radical generation.
• Experimental animal studies have shown that 5-FU, methotrexate, and cisplatin
are less effective at killing hypoxic cells than they are at killing normoxic tumor
cells
• Hypoxic tumor regions are frequently associated with a low pH that can also
diminish the activity of some chemotherapy agents.

28. Hypoxia and Tumor Progression
• Low oxygen condition play an important role in malignant progression of tumors
• Hypoxic cells limit the success in radiotherapy
• A clinical study in Germany in the 1990s showed a correlation between local control
in advanced carcinoma of the cervix treated by radiotherapy and oxygen-probe
measurements.

30. Hypoxia in clinical practice

31. HOW TO REDUCE HYPOXIA
Fractionation
Hyperbaric oxygen therapy
Cell sensitizers
Improving oxygenation of tumor by
Blood transfusion
Nicotinamide
Carbogen

32. Fractionation:
- Limiting damage to normal tissue
- Reoxygenation of hypoxic tumor cells
- Proportion of hypoxic cells lowers.
Hypoxia cell sensitizers
increase radiosensitivity of hypoxic but not oxic cells via free-radical formation
that mimics oxygen fixation of damage. Sensitizers are more metabolically
stable than O2, and thus able to diffuse into chronically hypoxic regions (≤200
μm).
eg. (Nitroimidazoles with high electron affinity)
- Misonidazole: more active, but toxic (peripheral neuropathy)
- Etanidazole: less toxic, but no benefit
- Nimorazole: less active, much less toxic (benefit in H&N cancer--Danish study)

36. Thank you