RADIOBIOLOGY: oxygen effect & reoxygenation

Oxygen Effect andReoxygenation

Presented by:Dr. Isha Jaiswal

Moderator: Prof. M.L.B Bhatt

Date:01 September 2014

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

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.

Mechanisms of oxygen effect

Indirect action of radiation

- 5

e-

X ray ray P+

OH

H OH-

H+

Ho

OHo

incident X ray photon

Fast e-

Ion radical

Free radical

Chemical changes

Biological changes

10-10 s

10-15 s

10-9 s

10-5 s

Hours, days, months, yrs, generations

Oxygen Acts At The Levels Of The Free Radicals.

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

The Oxygen fixation hypothesis.

Oxygen “fixes” (i.e. makes permanent) the damage produced by free radicals. The formation of RO2,an organic peroxide, repre-sents 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.

Oxygen Enhancement Ratio (OER)

The ratio of HYPOXIC to AEROBIC dosesneeded to achieve the SAME biological effects

Dose (Gy)

0 10 20 30

Su

rviv

ing

Fra

cti

on

0.01

0.1

1

AerobicHypoxic

1/e1/e

OER =D0 (hypoxic)

D0 (aerobic)= 3.22

D0D0

OER =

D0 (hypoxic)

D0 (aerobic)

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

1. Cellular Sensitivity

Resistant Cells : High OER

Sensitive Cells : Low OER

Radiation sensitive cells can be killed at lower dose. Thus, the OER is smaller.

The more resistant cells require large amounts of damage. Therefore, OER is more pronounced at higher dose.

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.

3. OER as a function of LET

4. Oxygen effect is time-dependent

Radiation Ion pairs Free Radicals

T = 10-10 sec T = 10-5 sec (0.01 msec)

Time delay is determined by lifetime of free radicals

For the oxygen effect to be observed, oxygen must be present during the radiation ex-posure or, to be precise, during or within microseconds after the radiation exposure.

Oxygen sensitization occurred with oxygen added as late as 5 milliseconds after irra-diation.

Oxygen acts at the level of the free radicals.

The free radicals break chemical bonds.

The extent of the damage depends on the presence or absence of oxygen.

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

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 from 1 to 100 mm Hg.

Many tissues are bordering hypoxic and contain a small proportion of cells that are radiobiologically hypoxic.

This is particularly true of, for example, the liver and skeletal muscles

• 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)

Types of hypoxia

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.

• 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.

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 highenough 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 oftumor.

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

The First Experimental Demonstration of hypoxia in tumor

Powers and Tolmach investigated radiation response of a solid subcutaneous lymphosarcoma in the mouse

The survival curve for this solid tumor clearly con-sists 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 sug-gests 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

• 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.

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.

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 oc-cur, the presence of hypoxic cells does not greatly influence the response of the tumor.

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.

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.

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.

Hypoxia in clinical practice

HOW TO REDUCE HYPOXIA

Fractionation

Hyperbaric oxygen therapy

Cell sensitizers

Improving oxygenation of tumor by

Blood transfusion

Nicotinamide

Carbogen

Fractionation: - Limiting damage to normal tissue- Reoxygenation of hypoxic tumor cells- Proportion of hypoxic cells lowers.

Hypoxia cell sensitizersincrease 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)

Thank you