This document discusses dose-response relationships for model normal tissues. It covers cell survival curve parameters, the linear-quadratic model, alpha/beta ratios, and dose-response curves derived from clonogenic and functional assays. Clonogenic assays examine cell survival and regrowth using tissues like skin, intestinal crypts, and testes. Functional assays measure outcomes like skin reactions, lung function, and spinal cord injury. Fractionation provides protection to late-responding normal tissues compared to tumors.

1. Dose-Response
Relationships for
Model Normal Tissues
Presented by- Dr. Rashmi Yadav
Moderated by- Dr. Ayush Garg

2. Introduction
• Dose-response relationships are important for prescribing a proper therapy
course
• Response is quantified as either increase of radiation effects in severity, or
frequency (% incidence), or both
• In vitro vs. in vivo experiments
• Different cells have different response based on their reproduction rate
(acute vs. late effects)
• With increasing radiation dose, radiation effects may increase in severity
(i.e., grade), in frequency (i.e., incidence), or both.

3. Cell Survival Curve Parameters
• D1 – initial slope (the dose required to
reduce the fraction of surviving cells to
37% of its previous value); D0 – final
slope
• Dq – quasi-threshold, the dose at which
the straight portion of the survival curve,
extrapolated backward, cuts the dose axis
drawn through a survival fraction of unity
• n – extrapolation number
• Radiosensitive cells are characterized by
curves with steep slope D0 and/or small
shoulder (low n)

4. Survival curves and LQ model
• Linear-quadratic model assumes there are two components to cell killing,
only two parameters
• An adequate representation of the data up to doses used as daily fractions in
clinical radiotherapy

5. α/𝜷 ratios
• If the dose-response relationship is adequately represented by LQ-model:
• The dose at which αD=𝜷D2 , or D= α/𝜷
• The α/𝜷 ratios can be inferred from multi-fraction experiments, assuming : –
each dose in fractionated regime produces the same effect
there is full repair of sub-lethal damage between fractions
there is no cell proliferation between fractions

6. • For the total dose D divided in n equal fractions of dose d
• If the reciprocal dose 1/dn is plotted against the dose per fraction d, the ratio
of the intercept/slope gives α/𝜷

7. Dose- response relationships
• With increasing radiation dose, radiation
effects may increase in severity (i.e.,
grade), in frequency (i.e., incidence), or
both.
• Curves are typically sigmoid (S)-shaped for
both tumor and normal cells (y axis is
“flipped” compared to cell survival curves)
• Therapeutic ratio (index) TR: tumor
response for a fixed level of a normal
tissue damage

8. Therapeutic ratio
• The ratio of the tumor response for a
fixed level of normal tissue damage has
been called either the therapeutic
ratio or the therapeutic index.
• The time factor is often employed to
manipulate the TR (hyper-fractionation
for sparing of late responding normal
tissues)
• Addition of a drug, a chemotherapy
agent, or a radiosensitizer may improve
the TR

9. Mechanisms of cell death after irradiation
• Cells die by various mechanism when they are exposed to radiation
1. Mitotic- linked cell death
2. Necrotic cell death
3. Apoptotic cell death
4. Autophagic cell death
5. Bystander induced cell death
6. Senescence

10. • The main target of radiation is cell’s DNA; single breaks are often reparable,
double breaks lethal
• Mitotic death – cells die attempting to divide, primarily due to asymmetric
chromosome aberrations; most common mechanism
• Apoptosis – programmed cell death; characterized by a predefined sequence
of events resulting in cell separation in apoptotic bodies-Cell shrinks,
chromatin condenses, cell breaks into fragments, no inflammation.
• Bystander (abscopal) effect – cells directly affected by radiation release
cytotoxic molecules inducing death in neighbouring cells

11. • Additional mechanisms under investigation: –
1. Autophagic: cell degradation of unnecessary or dysfunctional cellular
components through lysosomes
2. Necrotic: cell swells, leakage of membrane, inflammation
3. Entosis: cell death by invasion

12. Assays for dose-response relationships
• Clonogenic end points
• Depend directly on reproductive integrity of individual cells (cell survival)
• Cell regrowth in situ and by transplantation into another site
• Functional end points
• Reflect the minimum number of functional cells remaining in a tissue or
organ
• Dose-response can be inferred from multi-fraction experiments
• More pertinent to radiation therapy

13. Early and late responding tissues
• Observation: cells of different tissues demonstrate different response
rates to the same radiation dose
• Rapidly dividing self-renewing tissues respond early to the effects of
radiation; examples: skin, intestinal epithelium, bone-marrow
• Late-responding tissues: spinal cord, lung, kidney
• Early or late radiation response reflects different cell turnover rates

14. CLONOGENIC END POINTS

15. Skin
Colonies

16. Skin colonies
• Central area is given a test dose
and monitored for cell re-
growth
• Image showing nodule of mouse
skin regrowing from single
survival cell in treated area

17. • There are practical limits to the range
of doses
• Determine D0 =1.35 Gy from the single
dose curve
• Dq =3.4 Gy from the curve separation
• Values for D0 and Dq are similar to
those obtained in vitro

18. Crypt cells of the mouse
jejunum
• Intestinal epithelium is a classic example of a self renewing
system
• Crypt cells divide rapidly and replenish cells on the top of
villi
• Mice are given total body irradiation and are sacrificed
after 3.5 days
• Radiation effect is assessed based on the number of
regenerating crypts per circumference of the sectioned
jejunum

19. • A: Section of mouse jejunum taken 3.5 days after a total body dose in excess of
10 Gy. Note the shortened villi and the regenerating crypts.
• B: Regenerating crypts shown at a higher magnification.

20. -Survival curves for crypt cells in the mouse jejunum
exposed to single or multiple doses of gamma-rays (1–20
fractions). The score of radiation damage is the number
of surviving cells per circumference (i.e., the number of
regenerating crypts per circumference of the sectioned
jejunum). This quantity is plotted on a logarithmic scale
against radiation dose on a linear scale
-The D0 for the single-dose survival curve is about 1.3 Gy.
-The shoulder of the survival curve is very large. The
separation between the single-dose survival and two-
dose survival curves indicates that the Dq is 4 to 4.5 Gy.

21. Testes Stem Cells
A: Histology of normal testis.
B: Histology of testis 35 days after a dose of 9 Gy of
gamma-radiation. Some tubules are completely devoid
of spermatogenic epithelium and some are not.

22. Single- and split- dose survival
curves for spermatogenic stem
cells of the mouse testis. The D0
is about 1.68 Gy. The Dq, assessed
from the horizontal separation of
the single- and split-dose curves,
is about 2.7 Gy.

23. Kidney tubules
• Late responding tissue
• One kidney per mouse is irradiated and examined 60 weeks later
• Rate of response as time required for depletion of the epithelium after a single
dose of 14Gy: – 3 days in jejunum – 12 to 24 days in the skin – 30 days in the
seminiferous tubules of the testes – 300 days in the kidney tubules

24. A. Normal ,proximal tubules in contact with the capsule
B: Sixty weeks after irradiation with 13 Gy. Note normal proximal tubules and glomeruli amid
ghosts of de-epithelialized tubules

25. The time required for depletion of the epithelium after a single dose of
14 Gy is about
• 3 days in the jejunum
• 12 to 24 days in the skin
• 30 days in the seminiferous tubules of the testes
• 300 days in the kidney tubules.

26. Clonogenic End Points: Donor
Recipient Approach
• Systems in which cell survival is assessed by transplantation into
another site:
 Bone-marrow stem cells
Thyroid
Mammary gland cells

27. Cells Transplanted to Another Site
Bone Marrow Stem Cells
From the donor mouse, a cell suspension is
made of nucleated isologous bone marrow.
A known number of cells are injected into
recipient mice previously irradiated with a 9-
Gy total body dose. The spleen is removed
from each recipient mouse 9 or 10 days later,
and the number of nodules are counted.

28. • About 104 cells must be injected into a recipient animal to produce one spleen
colony because most of the cells in the nucleated isologous bone marrow are
fully differentiated cells and would never be capable of forming a colony.

29. Mammary and Thyroid Cells
• To generate a survival curve for mammary or thyroid gland cells in the rat, cells
may be irradiated in vivo before the gland is removed from donor animals and
treated with enzymes to obtain a monodispersed cell suspension. Known
numbers of cells are injected into the inguinal or interscapular white fat pads of
recipient animals.
• Under appropriate host conditions and grafted cell numbers, the injection of
mammary cells gives rise to mammary structures that are morphologically and
functionally normal. One such mammary structure may develop from a single
cell. By 3.5 weeks after the injection of mammary cells, positive growth is
indicated by alveolar units.

30. Clonogenic Assays:
Summary
• Radiosensitivity of a cell line is
determined by the curve shoulder
(Dq parameter)
• Significant variability observed

31. Functional End Points

32. Dose-response Curves For Functional
End Points
• Can be obtained on pig and rodent skin by assessing skin reaction
• For mouse lung system based on breathing rate, assess early and
late response
• Spinal cords of rats by observing myelopathy after local irradiation
complex system – various syndromes are similar to those described
in humans

33. Skin

34. Lung
• Early response (i.e., Pneumonitis) by 16-36 weeks,
• Late response (i.e., Fibrosis) by 52 weeks

35. Spinal Cord System
• Assess late damage caused by local irradiation of the spinal cords of rats
• First symptoms develop after 4 to 12 months
• Delayed injuries peak at 1 to 2 years post-irradiation
• The regimen of dose delivery has a strong effect on the resultant effect such
as the extend of necrosis, loss of functionality, etc.
• Obtain the information on the tolerance to radiation

36. • Dose-response relationship is steep
• Fractionation demonstrates dramatic sparing
• Latency decreases with increase in dose
• The region of cord irradiated

37. • The total volume of irradiated tissue has to have an effect on the
resultant injury
• Functional subunits (FSUs) in a spinal cord are arranged in linear
fashion therefore above a certain length of ~1cm the dependence is
very weak

38. INFERRING THE RATIO α/𝜷 FROM MULTIFRACTION
EXPERIMENTS IN NONCLONOGENIC SYSTEMS
• The dose–response relationship is represented adequately by the LQ formulation:
•S= e -αD-𝜷D2
• in which S is the fraction of cells surviving a dose, D, and α and 𝜷 are constants.
• Each dose in a fractionated regimen produces the same biologic effect.
• Full repair of sublethal damage takes place between dose fractions, but no cell
proliferation occurs.

39. SUMMARY
• The relationship between dose and incidence is sigmoid for both tumor control and normal tissue
damage.
• The ratio of tumor response to normal tis- sue damage is called the therapeutic ratio or therapeutic
index.
• The therapeutic index can be manipulated by dose fractionation or by the use of drugs that
preferentially increase tumor response.
• After irradiation, most cells die a mitotic death; that is, they die in attempting the next or a later
mitosis. In some tissues, cells die by apoptosis, which is a programmed cell death.
• Systems involving clonogenic end points (i.e., cell survival) for cells of normal tissues include some
in which cells regrow in situ and some in which cells are transplanted to another site.

40. • In situ regrowth techniques include skin colonies, crypts in the jejunum, testes stem cells,
and kidney tubules. Single dose experiments can yield the slope (D0) of the dose-
response curve over a range of high doses. Multifraction experiments allow the whole
dose-response curve to be reconstructed.
• Systems in which cell survival is assessed by transplantation into another site include bone
marrow stem cells, thyroid cells, and mammary cells.
• Dose–response curves for mammary and thyroid cells can be obtained by transplanting
them into fat pads of recipient animals.
• The radiosensitivity of cells from normal tissues varies widely. The width of the shoulder of
the curve is the principal variable. Jejunal crypt cells have a very large shoulder; bone
marrow stem cells have little, if any, shoulder. Most other cell types studied in clonogenic
assays fall in between.

41. Dose–response curves for functional end points, distinct from cell survival, can be
obtained as follows:
1. Pig skin and rodent skin—by measuring skin reactions.
2. Early and late response of the lung—by measuring breathing rate.
3. Spinal cord—by observing myelopathy
a. Paralysis develops after a latency of months to years.
b. Early lesions are limited to white matter; late delayed injury may have a vascular basis.
c. Spinal cord damage is very sensitive to fractionation—α/𝜷 of about 1.5 Gy.
d. Sublethal damage repair probably has “fast” and “slow” components.
e. If multiple fractions per day are used, the interfraction interval should be at least 6 to 8 hours.
f. Functional subunits are arranged serially like links in a chain.
g. For short lengths of cord, tolerance dose varies markedly with cord length irradiated.

42. • The shape of the dose–response relationship for functional end points, obtained from
multi-fraction experiments, is more pertinent to radiotherapy than clonogenic assays.
• The ratio α/𝜷 (the dose at which the linear and quadratic components of radiation
damage are equal) may be inferred from multifraction experiments in systems scoring
non-clonogenic end points.