Page 36 - 2014 Printable Abstract Book
P. 36
(S302) Small animal models for bystander studies with the microbeam. Manuela Buonanno; Gerhard
Randers-Pehrson; Guy Garty; Lubomir Smilenov; and David J. Brenner, Columbia University, New York, NY
Microbeams have been fundamental to investigate radiation-induced bystander effects in cell
cultures and 3-D systems. The next logical step is to develop and implement microbeam irradiation
protocols to study effects in living organisms. With the Columbia microbeam we can routinely study
targeted and non-targeted effects in living Caenorhabditis elegans, and a microfluidic worm clamp was
implemented for high throughput studies; on a single chip several living samples can be immobilized,
imaged and microbeam irradiated. Recently, we have developed a mouse ear model for bystander studies.
With an average thickness of 250 µm, the ear of a mouse can be used to investigate radiation-induced
bystander effects with the 3-MeV proton microbeam (LET 12.5 keV/µm) whose range is 134 µm. In our
current protocol, the ear of anesthetized mouse is flattened onto the underside of a flat plate of a custom-
made holder using gentle suction; the flattened mouse ear is then placed over the microbeam port and
cells along a line of the ear are irradiated with the proton microbeam with a selected diameter. At chosen
times after irradiation, mice are sacrificed and a punch of the ear is collected. Tissues are then fixed and
cut in 5-µm sections perpendicularly to the direction of the incident particle beam. The sections are then
analyzed for biological endpoints (i.e. formation of repair protein foci, apoptosis) as a function of the
distance from the irradiated line. Using γH2AX foci formation as endpoint assessed by
immunohistochemical analysis, we found that compared to control proton irradiation induced γH2AX foci
formation in ears of C57BL/6 mice. On microscopic cross section, the ear of a mouse consists of two full-
thickness layers of skin separated by a thin supporting skeleton of elastic cartilage: as expected, γH2AX
foci-positive keratinocytes were observed only in one of the two epidermal layers of the mouse ear. Cells
adjacent or in the epidermal layer opposite to the γH2AX positive region did not exhibit foci. Strikingly, a
higher number of cells than expected showed foci. These results suggest that microbeam proton
irradiation induced DNA damage in bystander cells in vivo. Ongoing experiments aim at investigating the
kinetics of gH2AX foci formation in microbeam irradiated ears as well as the molecular mechanisms
underlying these effects.
(S303) Widespread genomic change after exposure of epithelium to charged particles. Amy
1
1
3
3
2
Kronenberg ; Stacey Gauny ; Gianfranco Grossi ; Dmytro Grygoryev ; Anna Ohlrich ; and Mitchell S.
1
3
2
Turker, Lawrence Berkeley National Lab, Berkeley, CA ; Universita Federico II, Naples, Italy ; and Oregon
3
Health & Science University, Portland, OR
One of the most challenging issues for extended human space flight beyond low earth orbit is the
radiation exposure of crewmembers. Our long-standing interest in charged particle-induced mutations
can contribute to the understanding of the mechanisms underlying cancer risks for humans. Experiments
performed at the NASA Space Radiation Laboratory have characterized autosomal mutations at the Aprt
locus in mouse kidney epithelium exposed to charged particles in vitro or as part of a whole body exposure
to mice. In our hybrid mouse strain (C57BL6 x DBA/2) it is possible to observe all types of radiation-induced
mutations, from single basepair substitutions to whole chromosome loss. We combined our PCR-based
analyses of mutational spectra following exposure to protons, Si, Ti, or Fe ions with chromosome painting
to more fully characterize mutagenic events associated with these charged particle exposures in
apparently normal epithelium. Chromosome-scale events are particularly evident as radiation signature
mutations. These events occur shortly after exposure, as shown in our in vitro studies, but they persist in
34 | P a g e
Randers-Pehrson; Guy Garty; Lubomir Smilenov; and David J. Brenner, Columbia University, New York, NY
Microbeams have been fundamental to investigate radiation-induced bystander effects in cell
cultures and 3-D systems. The next logical step is to develop and implement microbeam irradiation
protocols to study effects in living organisms. With the Columbia microbeam we can routinely study
targeted and non-targeted effects in living Caenorhabditis elegans, and a microfluidic worm clamp was
implemented for high throughput studies; on a single chip several living samples can be immobilized,
imaged and microbeam irradiated. Recently, we have developed a mouse ear model for bystander studies.
With an average thickness of 250 µm, the ear of a mouse can be used to investigate radiation-induced
bystander effects with the 3-MeV proton microbeam (LET 12.5 keV/µm) whose range is 134 µm. In our
current protocol, the ear of anesthetized mouse is flattened onto the underside of a flat plate of a custom-
made holder using gentle suction; the flattened mouse ear is then placed over the microbeam port and
cells along a line of the ear are irradiated with the proton microbeam with a selected diameter. At chosen
times after irradiation, mice are sacrificed and a punch of the ear is collected. Tissues are then fixed and
cut in 5-µm sections perpendicularly to the direction of the incident particle beam. The sections are then
analyzed for biological endpoints (i.e. formation of repair protein foci, apoptosis) as a function of the
distance from the irradiated line. Using γH2AX foci formation as endpoint assessed by
immunohistochemical analysis, we found that compared to control proton irradiation induced γH2AX foci
formation in ears of C57BL/6 mice. On microscopic cross section, the ear of a mouse consists of two full-
thickness layers of skin separated by a thin supporting skeleton of elastic cartilage: as expected, γH2AX
foci-positive keratinocytes were observed only in one of the two epidermal layers of the mouse ear. Cells
adjacent or in the epidermal layer opposite to the γH2AX positive region did not exhibit foci. Strikingly, a
higher number of cells than expected showed foci. These results suggest that microbeam proton
irradiation induced DNA damage in bystander cells in vivo. Ongoing experiments aim at investigating the
kinetics of gH2AX foci formation in microbeam irradiated ears as well as the molecular mechanisms
underlying these effects.
(S303) Widespread genomic change after exposure of epithelium to charged particles. Amy
1
1
3
3
2
Kronenberg ; Stacey Gauny ; Gianfranco Grossi ; Dmytro Grygoryev ; Anna Ohlrich ; and Mitchell S.
1
3
2
Turker, Lawrence Berkeley National Lab, Berkeley, CA ; Universita Federico II, Naples, Italy ; and Oregon
3
Health & Science University, Portland, OR
One of the most challenging issues for extended human space flight beyond low earth orbit is the
radiation exposure of crewmembers. Our long-standing interest in charged particle-induced mutations
can contribute to the understanding of the mechanisms underlying cancer risks for humans. Experiments
performed at the NASA Space Radiation Laboratory have characterized autosomal mutations at the Aprt
locus in mouse kidney epithelium exposed to charged particles in vitro or as part of a whole body exposure
to mice. In our hybrid mouse strain (C57BL6 x DBA/2) it is possible to observe all types of radiation-induced
mutations, from single basepair substitutions to whole chromosome loss. We combined our PCR-based
analyses of mutational spectra following exposure to protons, Si, Ti, or Fe ions with chromosome painting
to more fully characterize mutagenic events associated with these charged particle exposures in
apparently normal epithelium. Chromosome-scale events are particularly evident as radiation signature
mutations. These events occur shortly after exposure, as shown in our in vitro studies, but they persist in
34 | P a g e
