The Oxygen Effect and Reoxygenation Radiobiology for the Radiologist, chapter 6, pg 91 - 111 The Oxygen Effect and Reoxygenation

The Nature of the Oxygen Effect

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

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

The Nature of the Oxygen Effect The oxygen enhancement ratio (OER) for various types of radiation Large & important Intermediate Absent

The Time at which Oxygen Acts

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

Mechanism of the Oxygen Effect

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

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

The Concentration of Oxygen Required

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

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

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

Chronic Hypoxia

Chronic Hypoxia Definition First described by Thromlison and Gray 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

Transverse section of tumor cord. Stroma Surrounded by intact tumor cells Central necrosis A typical tumor area in which necrosis is not far advanced.

Large areas of necrosis 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

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)

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

Acute Hypoxia

Acute Hypoxia Definition First postulated by Brown in 1980s 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

Diagram illustrating the difference between chronic and 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 Acute hypoxia Chronic Hypoxia Diagram illustrating the difference between chronic and acute hypoxia

The First Experimental Demonstration of Hypoxic cells in a tumor

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

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

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

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

Proportion of Hypoxic cells in Various Animal Tumors

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

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

Reoxygenation

Reoxygenation Van Putten and Kallman Result: 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.

Significant of fractionation 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

Time Sequence of Reoxygenation

Time Sequence of Reoxygenation 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 Immediately after irradiation 100% of viable cells are hypoxic By 6 hours, percentage of hypoxic cells has fallen to a close value to the preirradiation level

The extent and rapidity of reoxygenation is extremely variable and impossible to predict △ Mouse osteosarcoma ● mouse fibrosarcoma Cell type Reoxygenation Mammary carcinoma Rapid & well Rat sarcoma Two waves Osteosarcoma Not all cells & slow Fibrosarcoma Rapid but not complete ▲ Rat sarcoma ○ Mouse mammary carcinoma The proportion of hypoxic cells as a function of time after irradiation with a large dose for 5 transplanted tumors in experimental animals

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

The Important of Reoxygenation in Radiotherapy

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

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

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

Hypoxia and Tumor Progression

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

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

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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