Immune System Response to Irradiation of Cells by Alpha Particles

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Immune System Response to Irradiation of Cells
by Alpha Particles
Brian Camley
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The Problem
Radon is a colorless, odorless, radioactive gas
that is believed to cause 10 of lung
cancers Radon kills an estimated 18,000 to 25,000
people in the US each year The estimate is based
on the linear, no-threshold model which states
that the cancer risk is simply proportional to
total exposure
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The Problem (continued)
The action level for radon mitigation is based,
in part, on the linear model. If the linear
model is wrong We could be wasting billions of
dollars each year on unnecessary mitigation
or There might
be thousands of needless deaths each year
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Where does the linear model come from?

• Studies on uranium miners
• Epidemiological studies
• Microscopic studies of damage to cells by
  alpha particles

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The linear model
National Council on Radiation Protection and
Measurements (NCRP), 1984a, Evaluation of
occupational and environmental exposures to radon
and radon daughters in the United States Report
78, 204
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Is the linear model right?

• Recent microscopic studies suggest the damage to
  cells is proportionally higher at low doses
• Some researchers have suggested that low doses of
  alpha particles stimulate the immune system and
  thus lower risk

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What should be done?
We need experiments which measure damage to
tissue and immune response simultaneously
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Outline

• Background on damage caused by alpha particles
• Background on the immune system
• The experimental work
• Results and Discussion
• Summary

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How does radon damage lungs?
Radon gas produces radon decay products. The
decay products are inhaled. Radon decay products
produce energetic (6-7 MeV) alpha particles which
hit and damage lung cells
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How does radon damage cells?
Alpha particles typically can pass through about
6 cells. The alpha particle will either hit the
nucleus or the cytoplasm. 1) If the alpha
particle hits the nucleus, it can directly
interact with DNA and cause mutations. Sometimes
these mutations will become cancerous.
Nucleus
2) If the alpha particle hits the cytoplasm, it
increases the number of ions and free radicals,
particularly reactive oxygen, within the cell.
Recent studies (Li-Jun Wu et al, 1999) have
indicated that this is more likely to cause
cancer than a direct hit on the nucleus!
Put picture here
Cytoplasm
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How does the immune system respond?

• T cells must be activated for the immune system
  to respond.
• The outside of T-cells have two types of
  receptors that must be engaged to activate the
  cell.
• Antigen in MHC
• Costimulation signal B7 or Fas (danger)

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Cell Growth
L.12.10 mouse leukemia cells were grown in a
typical RPMI media. This required Incubating
the cells at 37 C Cutting back the cells
twice-weekly to prevent them from becoming
overcrowded Refreshing the media to prevent it
from becoming acidic. Counting the cells with a
hemacytometer to determine their density
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The experiment
17,000 picocurie RaNO3 source alpha particle
emitter
4 million cells L.12.10
5 mm
10, 20, 30, and 40 minute exposure times
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Calculating the Dosage Point Source
Number of alpha particles that hit well plate
(Number leaving source) (percent surface area
of well plate)
q
For ? 49.4o Number hitting 17.5 (Number
leaving)
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Calculating Dosage
Numerical Model
Point Source Result Spread Out Source Result
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Dosages
10 minutes 105,000 cells hit (2.6
) 20 minutes 210,000 cells hit (5.2
) 30 minutes 315,000 cells hit (7.8
) 40 minutes 420,000 cells hit (10.4
)
Numerical calculations were in good agreement
with values measured using alpha track foils
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Flow Cytometry

• Cells are stained with different fluorescent dyes
• The dyes attach themselves to different parts of
  the cell some to reactive oxygen and some to
  the costimulatory molecules
• The cells are sent (one at a time) past a laser
  and the resulting light is analyzed

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Flow Cytometry

Sheath Fluid
Laser Beam
Fluorescent signals
Individual cells
Side Scatter
Forward Scatter
The fluorescent signals give information on
presence of reactive oxygen and B7.1. The ratio
of forward scatter to side scatter gives
information of whether a cell is living or dead
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Controlling Error
Prior studies have shown that even irradiating
the cell growth media increases the reactive
oxygen of the cells. In addition, alpha
particles cannot go through more than about 6
layers of cells. This problem was removed by
centrifuging the cells into about four layers on
the bottom of a well plate. It is important to
note that this procedure stressed the cells, and
may have increased the levels of H2O2 and B7.1
but it should do so to all cells. A control
group that had the same procedure except no
radiation was used to monitor this.
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The Results
Initial results on exposure of mouse leukemia
cells to alpha particles
Cells die at a faster rate with alpha particle
exposure
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The Results
Mouse Leukemia Cells (L.12.10)
Reactive Oxygen - Damage
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The results for reactive oxygen are qualitatively
similar to 2001 results by Zhou et al showing
mutations/survivors as a function of percentage
of cells irradiated.
Measured Mutations
Expected from linear model
There is a rapid rise and then saturation
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The results for reactive oxygen are qualitatively
similar to those from T. Hei et al showing
mutations/survivors as a function of percentage
of cells irradiated.
Measured Mutations
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The Results
Mouse Leukemia Cells (L.12.10)
B7 Immune Response
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What does this mean for the linear theory?
The linear theory does not predict the amount of
damage it predicts the risk from that
damage. How would you calculate risk? Risk goes
up with greater damage, but should go down as the
immune system responds.
Combining B7.1 and Reactive Oxygen Risk
H2O2 levels
B7.1 levels
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The Linear Theory
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Explaining Reactive Oxygen
How do we make sense of these results? -
Saturation? - Other effects?
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The Stupid Simulation
You can use a bystander effect to explain these
results, by way of a very simple simulation.
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H2O2
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H2O2
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H2O2
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H2O2
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H2O2
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H2O2
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H2O2
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H2O2
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H2O2
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H2O2
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To agree with the results that saturation occurs
near 10 or 20 exposure a bystander effect with
an effective radius of 3 cells is required.
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Conclusion

• Exposure to alpha particles does result in a
  disproportionately high level of mutagenic damage
  at lower levels.
• This is due to the bystander effect and
  saturation, and not a particular property of
  alpha particles.
• B7.1 levels do not rise significantly and may in
  fact be suppressed by exposure to alpha
  particles. The immune system is suppressed!
• Combining H2O2 and B7.1 levels predicts a risk of
  cancer that corresponds with the linear theory.
• Computer modeling predicts that an alpha impact
  on a cell affects all cells within a three-cell
  radius.

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Acknowledgements
I would like to thank the following people for
their moral support, training, supervision, and
(to sum it up) their unfailing patience in
dealing with me (in no particular order)
Christa Ford trained me in the beginning
techniques in a biology lab Karen Newell
introduced me to the concept of B7 and immune
system response Jim Burkhart and Bob Camley
provided the radium samples and helpful
discussions Jeff Rogers and Lisa
Villalobos-Menuey ran the flow cytometer, as I
was not allowed to for insurance reasons Karen
Newell also allowed me to work in her laboratory
at the University of Colorado, Colorado Springs
Biology Department