radiobiology oxygen effect and hypoxia

1. and
Hypoxia
DR SAILENDRA
SENIOR RESIDENT
DEPT OF RADIOTHERAPY
MAULANA AZAD MEDICAL COLLEGE
Oxygen
effect

2. Road map
• What is oxygen effect ?
• What is the mechanism ?
• What is the timing of action of oxygen ?
• What is the concentration of oxygen ?
• Different types of hypoxia ?
• What is reoxygenation ?
• Biology of tumour hypoxia.
• Modes to overcome hypoxia effect.

3. history
• The oxygen effect was observed as early as
1912 in Germany by Swartz, who noted that
the skin reaction produced on his forearm by a
radium applicator was reduced if the
applicator was pressed hard onto the skin.
• 1921,it had been noted by Holthusen that
Ascaris eggs were relatively resistant to
radiation in the absence of oxygen
• In England in the 1930s, Mottram explored the
question of oxygen in detail.

4. Oxygen effect
OER IS LOW AT LOWER DOSES AND A BIT HIGH AT HIGHER DOSES

5. THE NATURE OF THE OXYGEN
EFFECT
oxygen enhancement ratio(OER)
The ratio of doses administered under hypoxic to aerated
conditions needed to achieve the same biologic effect.
Oxygen enhancement ratio =
For sparsely ionizing radiations, such as x-rays and γ-rays, the
OER at high doses has a value of between 2.5 and 3.5.
Radiation dose
in Hypoxia
Radiation dose
In Air

7. summary
The oxygen effect is large and important in the case
of sparsely ionizing radiations, such as x-rays.
Absent for densely ionizing radiations, such as
α-particles.
Intermediate value for fast neutrons.

8. THE TIME AT WHICH OXYGEN ACTS AND THE
MECHANISM OF THE OXYGEN EFFECT
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.
Howard-Flanders and Moore, 1958; Michael et al., 1973

9. The DNA radical are deactivated to its
reduced form through reaction with a
sulfhydryl (SH) group.

10. • The damage produced by free radicals in
DNA can be repaired under hypoxia but may
be “fixed” (made permanent and
irreparable) if molecular oxygen is
available.This is known as the oxygen
fixation hypothesis.

11. THE CONCENTRATION OF
OXYGEN REQUIRED
• If the radiosensitivity
under extremely anoxic
conditions is assigned a
value of 1, the relative
radiosensitivity is about 3
under well-oxygenated
conditions.
• Maximum change of
sensitivity occurs at the
oxygen tension 30 mm Hg.
• A further increase of
oxygen content has little
further effect.

12. CHRONIC AND ACUTE HYPOXIA
Chronic
hypoxia
Limited
diffusion
distance of
oxygen through
tissue
Acute
hypoxia
Temporary closing of a
tumor blood vessel owing to
the malformed vasculature
of the tumor
• Lacks smooth muscle
• Incomplete endothelial lining
• Incomplete basement membrane

14. Chronic Hypoxia
• Chronic hypoxia was first described by
Thomlinson and Gray in 1955.
• By viewing histological sections of fresh
specimens of human bronchial carcinomas.

15. • Thickness of
the sheath of
viable tumor
cells remains
essentially
constant.
From Thomlinson RH, Gray LH

16. • They calculate the distance to which oxygen
could diffuse in respiring tissue and came
up with a distance of about 150μm which
was close enough to the thickness of viable
tumor cords on their histologic sections.

17. From Thomlinson RH, Gray LH, Br J Cancer. 1955;9:539–549

19. Acute Hypoxia
• Postulated in the early 1980s by Martin Brown.
• Temporary closing or blockage of a particular
blood vessel.
• Tumor blood vessels open and close in a
random fashion, so that different regions of the
tumor become hypoxic intermittently.
• That is why fractionated radiotherapy can
overcome acute hypoxia.

20. EXPERIMENTAL DEMONSTRATION OF
HYPOXIC CELLS IN A TUMOR
• If the shallow component
of the curve is
extrapolated backward to
cut the surviving-fraction
axis, it does so at a
survival level of about
1%.
• From this, it may be
inferred that about 1% of
the clonogenic cells in the
tumor were deficient in
oxygen.
Powers WE, Tolmach LJ.survival curve for mouse lymphosarcoma cells irradiated in vivo.
Nature. 1963;197:710–711
D0 =1.1 Gy
D0 =2.6 Gy

21. TECHNIQUES TO MEASURE
TUMOR OXYGENATION
• Gold standard
• Invasive
Oxygen Probe
Measurements
• Pimonidazole
• Noninvasive
• IHC from biopsy specimen for carbonic anhydrase
IX (CA9) and HIF-1
• Differentiate between viable and necrotic tissue
Markers of
Hypoxia

22. pretreatment frozen biopsy from a patient with
carcinoma of the cervix
• Green – pimonidazole (hypoxic cells)
• Red - nuclei that express the HIF-1 (regions with low
oxygen tension)
• Blue - blood vessels
Cancer Res. 2005;65:7259–7266

23. REOXYGENATION
• Van Putten and Kallman determined the proportion of
hypoxic cells in a transplantable sarcoma in the mouse.
• Hypoxic cells in the untreated tumor was about 14%.
• When groups of tumors were exposed to five daily doses
of 1.9 Gy delivered Monday through Friday, the
proportion of hypoxic cells was determined on the
following Monday to be 18%.
• In another experiment, four daily fractions were given
Monday through Thursday, and the proportion of
hypoxic cells measured the following day, Friday, was
found to be 14%.

24. summary
• Proportion of hypoxic cells in the tumor is
about the same at the end of a fractionated
radiotherapy regimen.
• This phenomenon, by which hypoxic cells
become oxygenated after a dose of radiation, is
termed Reoxygenation.
• If reoxygenation is efficient between dose
fractions, the presence of hypoxic cells does not
have a significant effect on the outcome of a
multifraction regimen.

26. MECHANISM OF
REOXYGENATION
• 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.
• This takes several days.
• It overcomes chronic hypoxia
• Other mechanism is reopening of the blood
vessels those were temporarily closed causing
acute hypoxia.It occurs within hours of
irradiation.

27. HYPOXIA AND
CHEMORESISTANCE
• Decreased free-radical generation.
• Associated with a low pH that can also
diminish the activity of some chemotherapy
agents.

28. HYPOXIA AND TUMOR
PROGRESSION
• 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.
• Specifically, patients in whom the probe
measurements indicated pO2s greater than 10
mm Hg did better than those with pO2s less
than 10 mm Hg.
• This suggested that the presence of hypoxic
cells limited the success of radiotherapy.

29. • Studies carried out in the United States on
patients receiving radiotherapy for soft
tissue sarcoma highlighted the correlation
between tumor oxygenation and the
frequency of distant metastases.
• 70% metastasis pO2s < 10 mm Hg
• 35% metastasis pO2s > 10 mm Hg.

30. RADIORESISTANCE
CHEMORESISTANCE
AGGRESSIVE
BEHAVIOUR

31. BIOLOGY OF TUMOUR HYPOXIA
HYPOXIA-INDUCIBLE FACTOR
• Hypoxia-inducible factors (HIFs) are transcription
factors that facilitate both oxygen delivery and
adaptation to oxygen deprivation by regulating the
expression of genes that are involved in many
cellular processes including glucose uptake and
metabolism, angiogenesis,erythropoiesis, cell
proliferation, and apoptosis.
• It has an α-sub unit and a β-sub unit.
• Three HIFs (HIF-1,-2, and -3) have been identified.

32. OXYGEN PRESENT
OXYGEN ABSENT
Angiogenesis
Erythropoiesis
Tissue remodeling
Glycolysis
MECHANISM OF ACTION OF HIF

33. Cancer Mutations that Activate
Hypoxia-Inducible Factor
• VHL and PTEN mutation can lead to
activation of HIF leading to multiple tumour
syndromes like MEN,renal cell
carcinoma,retinal hemangioblastoma,CNS
neoplasms,pancreatic tumours etcs.

34. Roles of Hypoxia Inducible Factor in Tumors
Angiogenesis By activating VEGF-A
Tumor
Metabolism
shift glucose metabolism from an oxidative to
glycolytic pathway
Tumor
Metastasis
promotes metastasis through the transcriptional regulation
of key factors such as E-cadherin, lysyl oxidase, and CXCR4
that govern cell adhesion,extracellular matrix formation,
and cell migration.
Radiotherapy
HIF-1 deficient tumors are more
sensitive to radiation compared to
wildtype tumors.

35. UNFOLDED PROTEIN
RESPONSE
• Stress -----------accumulation of unfolded
proteins-----------cell death
• IRE 1--------activate XBP1(transcription
factor)----------induce chaperon synthesis-------
-folding of proteins----------cell survive----------
tumourigenesis
• PERK(translation inhibitor)--------activated
due to hypoxic stress----------inhibit translation
or protein synthesis---------misfolded protein
synthesis stops---------cell survive----------
tumourigenesis

36. I
R
E
ACTIVATION
OF XBP-1
INDUCE
CHAPERON
SYNTHESIS
FOLDING OF
PROTEINS
CELL
SURV
IVES
PERK
INHIBITION OF
PROTEIN
SYNTHESIS
MISFOLDED
PROTEIN
SYNTHESIS
STOPS
CELL
SURVIVES
T
U
M
O
U
R
I
G
E
N
E
S
I
S
UNFOLDED PROTEIN RESPONSE

38. RADIOSENSITIZING HYPOXIC
CELLS
• Hyperbaric oxygen
• Chemical radiosensitizers
• Hypoxic cytotoxins.
• Blood transfusion

39. HYPERBARIC OXYGEN
• Patients were sealed in chambers filled with
pure oxygen raised to a pressure of 3
atmospheres
• Patient compliance was a problem
• The clinical trials involved small numbers of
patients and unconventional fractionation
schemes. (mostly large fractions and short
duration)
• Risk of fire

40. • The largest multicenter trials performed by the
Medical Research Council in the United
Kingdom
• Showed a significant benefit both in local
control and in survival for patients with
carcinoma of the uterine cervix and advanced
head and neck cancer.
• The trials showed a 6.6% improvement in local
control
• Increase in late normal tissue damage.

41. • Hyperbaric oxygen lost its popularity
• Because smoking can decrease tumor
oxygenation, it is clearly advisable for
patients to give up smoking,at least during
radiotherapy

42. Properties essential for hypoxic cell
sensitizer
Selectively sensitize hypoxic cells
Chemically stable
Highly soluble in water or lipids
Should be effective at the relatively low daily doses.

43. MISONIDAZOLE
• Misonidazole has a dramatic effect on
tumors in experimental animals.
• If x-rays(single dose) are used alone, the
dose required to control half of the mouse
mammary tumors is 43.8 Gy. This falls to
24.1 Gy if the radiation is delivered 30
minutes after the administration of
misonidazole (1 mg/g body weight).This
corresponds to an enhancement ratio of 1.8.

44. Sheldon PW, Foster JL, Fowler JF. Radiosensitization of C3H
mouse mammary tumours by a 2-nitroimidazole drug. Br J Cancer. 1974;30:560–565

45. • This dramatic effect is only seen in single-
dose treatments,in contrast to the
multifraction regimens common in
conventional radiotherapy.
• More than 20 trials conducted by RTOG,but
none yielded a statistically significant
advantage for misonidazole.

46. • Danish head and neck cancer trial of
misonidazole.
• Significant improvement of tumor control by
radiotherapy only for males with tumors of the
pharynx and depended on hemoglobin status.
Data from Dr. Jens Overgaard

47. • Dose limiting toxicity of misonidazole was
found to be peripheral neuropathy that
progressed to central nervous system
toxicity on continuous use.

48. Metronidazole
↓
Misonidazole: more active, toxic
benefit in subgroups
↓
Etanidazole: less toxic, no benefit
↓
Nimorazole: less active, much less toxic
benefit in head and neck cancer

49. Overgaard’s
Meta-analysis
• Overgaard and colleagues performed a meta-
analysis.
• They identified 10,602 patients treated in 82
randomized clinical trials involving hyperbaric
oxygen, chemical sensitizers, carbogen
breathing, or blood transfusions.
• Overall, local tumor control was improved by
4.6%, survival by 2.8%, and the complication
rate increased by only 0.6%, which was not
statistically significant.

50. • Head and neck tumors showed the greatest
benefit.
• It was also concluded that the problem of
hypoxia may be marginal in most
adenocarcinomas and most important in
squamous cell carcinomas.

51. HYPOXIC CYTOTOXINS
1. Quinone antibiotics e.g Mitomycin C
2. Nitroaromatic compounds....high toxicity
3. Benzotriazine di-N-oxides...tirapazamine
4. Dinitrobenzamide modified nitrogen
mustard
5. 2-nitroimidazole attached to dibromo
isophosphoramide

52. TIRAPAZAMINE
Tumor volume as a function of time after various
treatments of an SCCVII transplantable mouse
carcinoma
Int J Radiat Oncol Biol Phys. 1991;20:457–461

55. There were no significant differences in Overall
survival, failure-free survival, time to locoregional
failure, or quality of life.

56. toxicitY
• Nausea and vomiting
• Diarrhea
• Weight loss
• Skin rash
• Muscle cramps
• Tinnitus ,acute reversible hearing loss
• Visual disturbances
• Cardiac ischemia and transient loss of
consciousness.
• Grade 3 and 4 neutropenia was reported in 4 of 39
patients treated with TPZ at 159 mg/m 2 three
times a week for 12 doses with radiotherapy for
head and neck cancers.

57. TO REMEMBER…..
• Hypoxia not only makes tumours
radioresistant but aggressive too.
• Misonidazole and Tirapazamine are two drugs
that are effective against hypoxic cells.
• OER is more marked in sparsely ionising
elements like x-rays and gamma rays.
• Chronic hypoxia is due to decreased Oxygen
diffusion.
• Acute hypoxia is due to temporary vasospasm.