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Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstracts
_____________________________________________________________________
Title:
Thresholds
for Radiation-Induced Mutations and Neoplastic Transformation
Could Arise from Apoptosis and Error-Free Repair.
Authors:
B.R. Scotta, Y. Tesfaigzia, J. Adena,
H. Schöllnbergerb, and D. Walkera.
Institutions:
aLovelace Respiratory Research Institute, bUniversity
of Salzburg.
In
June 2001, the National Council on Radiation Protection and
Measurements published Report No. 136, “Evaluation of
the linear-nonthreshold dose-response model for ionizing radiation.”
The report concluded that the linear-nonthreshold (LNT) model
was valid for characterizing low-dose radiation risks. Further,
the report states at the end of the Executive Summary: “the
probability of effects at very low doses such as are received
from natural background … is so small that it may never
be possible to prove or disprove the validity of the linear-nonthreshold
assumption.” In Klaus Becker’s review of the report
(Health Phys. 82, No. 2, 257-258,
2002), he strongly criticized the conclusion regarding the validity
of the LNT hypothesis. In this presentation, we discuss strong
evidence that relates to our research against the validity of
the LNT model.
Our
research has focused on modeling stochastic effects induced
in cells by low doses of genotoxicant agents. Using genomic
instability state (GIST) models, we can predict induced mutation,
cell killing, and neoplastic transformation frequencies after
low doses. Our most advanced GIST model, NEOTRANS2, has recently
been revised in light of new data demonstrating large thresholds
for cancer induction after low dose rate, low-LET irradiation
(Rossi, H., and Zaider, M., Radiat. Environ. Biophys.
36:85-88, 1997; Yamamato, O. et al.,
Int. J. Radiat. Biol. 73: 535-541,
1998; Kondo, S. J., Nucl. Sci. Tech 36:1-9,
1999) and new research results related to natural protection
against stochastic effects of irradiation (Barcellos-Hoff, M.H.,
and Brooks, A.L., Radiat. Res. 156:618-627,
2001; Tanooka, H. in Biological Effects of Low Dose Radiation,
Elsevier Science B.V., Amsterdam, pp. 155-160, 2000).
The
NEOTRANS2 model includes both hypersensitive (Figure 1) and
resistant cells (not shown). Only the hypersensitive fraction,
f1, is assumed affected by low radiation doses. Resistant cells
are assumed affected by moderate and high doses.
Like
its predecessor, NEOTRANS1, NEOTRANS2 is based on the premise
that ionizing radiation induces differing classes of genomic
instability: transient minor instability (TMI), transient problematic
instability (TPI), and persistent problematic instability (PPI)
in resistant cells; and TPI and PPI in hypersensitive cells
that are considered to initially possess normal minor instability
(NMI) due to an existing genomic abnormality. Dr. Dale Walker’s
recent data related to in vitro exposure of mammalian cells
to a genotoxic chemical supports the postulated instability
classes.
The
variable c represents the dose rate. The parameters a1
accounts for target cell genomic sensitivity to damage induction;
m1 for the commitment of damaged cells to the error-free repair
pathway; h1 for cells that undergo nonlethal misrepair
leading to mutations; j1 for cell commitment to the
apoptosis pathway; and k1c for cells undergoing the
necrotic mode of cell death (due to additional damage).
Figure
1. NEOTRANS2 model, hypersensitive cells only. Abbreviations
are defined in the text.
Although both apoptosis and necrotic cell death pathways are
included in NEOTRANS2, at very low radiation doses, only apoptosis
is considered important and then only for hypersensitive cells.
With NEOTRANS2, a common initial damage pathway relates to the
induction of genomic instability (transient). This pathway places
the hypersensitive cells at risk for undergoing apoptosis, necrotic
death (via additional damage production), gene mutations, and
subsequent neoplastic transformation. The error-free repair
pathway allows for protection from such effects. Apoptosis also
protects the cell community from mutations and neoplastic transformations.
Mutations (PPI cells) arise via non-lethal misrepair of genomic
damage and lock in persistent genomic instability, rendering
cells more susceptible to neoplastic transformation. This transformation
arises in progeny of the mutated cells (a result of single or
multiple stochastic changes in the genome). Different mutations
are included in the PPI cells so that no particular mutation
type is considered responsible for neoplastic transformation.
Neoplastic transformation is assumed to arise spontaneously
from heightened and lasting genomic instability. Cells with
PPI are assumed more likely to undergo additional mutations,
locking in even higher levels of genomic instability.
A
very small proportion (stochastic quantity), T0, of cells at
risk is assumed to already be committed to spontaneous neoplastic
transformation, because of previous events during their life
history. These cells are treated as being at risk of undergoing
apoptosis via a bystander effect of irradiating the other cells
not already committed to neoplastic transformation. Initially,
it was assumed that all T0 cells are eliminated via the bystander
effect for apoptosis; however, we have now relaxed this requirement
and consider that only some fraction of the T0 cells is killed.
Presently, it is assumed that this fraction is independent of
radiation dose but may depend on the type of radiation, the
spatial distribution of the dose, and biological characteristics
of the target cell community.
In
the current version of NEOTRANS2, the misrepair pathway is assumed
to apply only for a dose rate in excess of a critical value
c*. This modeling change is based on the observation by researchers
that chronically administered (low dose rate) low-LET irradiation
appears to induce cancer only after very large radiation doses
(Rossi, H., and Zaider, M., Radiat. Environ. Biophys.
36:85-88, 1997; Yamamato, O. et al.,
Int. J. Radiat. Biol. 73:535-541,
1998; Kondo, S.J., Nucl. Sci. Tech 36:1-9,
1999; Tokarskaya, Z. B. et al., submitted to Health
Phys.).
Research
results indicate that the shape of the dose-response curve (for
doses in excess of background radiation) for neoplastic transformation
at low radiation doses cannot be of the LNT type if any of the
following occurs within the low-dose region of interest: (1)
no repair errors (e.g., after low-dose-rate, low-dose, low-LET
irradiation); (2) removal via apoptosis of all cells likely
to undergo misrepair (e.g., after high-LET irradiation); and
(3) repair induction (activation) above a damage threshold.
In cases 1 and 2, a dose threshold would be expected for induced
mutations and neoplastic transformation.
David
Hoel and P. Li (Health Phys. 75 No.
3, 241-249, 1998) have demonstrated that use of a threshold–type,
dose-response model leads to better characterization of both
the leukemia incidence and mortality data for atomic bomb survivors
than use of the LNT model. In addition, Z.B. Tokarskaya and
colleagues (Health Phys. 73, No. 6,
899-905, 1997) reported a threshold dose close to 1 Gy for lung
cancer induction by alpha radiation, based on Mayak workers
that inhaled plutonium-239. R. E. Rowland has reported an even
larger threshold (Radioprotection 32,
C1-331 – C1-338, 1997) for bone cancer induction by alpha
radiation for radium dial painters. Thus, the adoption of the
LNT model by the NCRP for low-dose risk assessment, as indicated
in NCRP Report 136 (also in NCRP Report 135, 2001),
is likely to be viewed as unfortunate by those who are concerned
about the lack of use of science-based risks estimates
for evaluating potential harm to humans from exposure to low
doses of ionizing radiation. (Research supported by the U.S.
Department of Energy, Offices of Science and Environmental Management).
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Biologically Based Analysis of Lung
Cancer Incidence in a Large Canadian Occupational Cohort with
Low-LET Low-dose Radiation Exposure, and Comparison with Japanese
Atomic Bomb Survivors.
Authors:
W. D. Hazelton, D. Krewski, S. H. Moolgavkar.
Lung cancer incidence is analyzed in a large Canadian National
Dose Registry (CNDR) cohort with individual annual dosimetry
for low-dose occupational exposure to gamma and tritium radiation
using several types of multistage models. The primary analysis
utilizes the two-stage clonal expansion model (TSCE), with sensitivity
analyses using extensions of this model incorporating additional
stages. Characteristic and distinct temporal patterns of risk
are found for dose-response affecting early, middle, or late
stages of carcinogenesis, e.g. initiation with one or more stages,
clonal expansion, or malignant conversion. Fixed lag or lag
distributions are used to model time from first malignant cell
to incidence. Background rates are analyzed by gender, job classification
and birth cohort. Lacking individual smoking data, surrogate
doses based on US annual per capita cigarette consumption appear
to account for much of the birth cohort effect. Males, with
mean cumulative exposure for gamma and tritium of 11.5 mSv and
322 incident lung cancer cases have a significant dose-response
with 33 cases attributable to radiation. Female dose-response,
with mean cumulative exposure of 1.7 mSv and 78 incident cases,
appears similar but is not statistically significant. Findings
for males include an inverse-dose-rate effect (increased risk
with protraction of a given dose) and dose-response effects
on initiation, promotion and malignant conversion, although
the effect on initiation is not statistically significant. The
excess relative risk (ERR) and excess absolute risk (EAR) depend
on age at exposure, duration, dose, and age at follow-up. The
ERR increases with dose, tapering off at higher doses, making
a plot of ERR against dose concave-downward, similar to apparent
low-dose results seen below 1 Sv for solid tumor mortality of
atomic bomb survivors. The concave-downward trend of ERR and
the inverse-dose-rate effect are both counter to prevailing
beliefs about effects of low-LET ionizing radiation. The dose-response
estimated from the Canadian data is consistent with the dose-response
seen in the A-bomb survivors’ data when account is taken
of the virtually instantaneous exposure in the latter cohort.
__________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Do Low Dose-rate Bystander Effects Influence
Domestic Radon Risks?
Authors:
D.J. Brenner † and R.K. Sachs ‡.
Institutions:
† Center for Radiological Research, Columbia
University ‡ Department of Mathematics, University of
California.
Radon risks derive from exposure of bronchio-epithelial cells
to high-LET alpha particles. Alpha particle exposure can result
in bystander effects, where irradiated cells emit signals resulting
in damage to nearby unirradiated bystander cells. This can result
in non-linear dose-response relations, and inverse dose-rate
effects. Domestic radon risk estimates are currently extrapolated
from miner data which are at both higher doses and higher dose
rates, so bystander effects on unhit cells could play a large
role the extrapolation of risks from mines to homes. We have
therefore extended an earlier quantitative mechanistic model
of bystander effects to include protracted exposure, with the
aim of quantifying the significance of the bystander effect
for very prolonged exposures.
A
model of high-LET bystander effects, originally developed to
analyze oncogenic transformation in vitro, is extended to low
dose rates. The model considers radiation response as a superposition
of bystander and linear direct effects. It attributes bystander
effects to a small subpopulation of hypersensitive cells, with
the bystander contribution dominating the direct contribution
at very low acute doses but saturating as the dose increases.
Inverse dose-rate effects are attributed to replenishment of
the hypersensitive subpopulation during prolonged irradiation.
The
model was fitted to dose- and dose-rate dependent radon-exposed
miner data, the results suggesting that one directly-hit target
bronchio-epithelial cell can send bystander signals to about
50 neighboring target cells. The model suggests that a naïve
linear extrapolation of radon miner data to low doses, without
accounting for dose rate, would result in an underestimation
of domestic radon risks by about a factor of four, a value comparable
to the empirical estimate applied in the recent BEIR-VI report
on radon risk estimation.
Bystander
effects represent a plausible quantitative and mechanistic explanation
of inverse dose-rate effects by high-LET radiation, resulting
in non-linear dose-response relations, and a complex interplay
between the effects of dose and exposure time. The model presented
here provides a potential mechanistic underpinning for the empirical
exposure-time correction factors applied in the recent BEIR-VI
for domestic radon risk estimation.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title:
Low Dose, Risk, Decisions and Risk Communication.
Authors:
James Flynn, Donald MacGregor and Paul Slovic.
Institutions:
Decision Research.
Issues of low dose radiation exposure generate a varied range
of responses from the public. Social issues such as historical
and popular portrayals of radiation play a role in the formation
of attitudes, helping to shape the overall context in which
events such as nuclear facility closure, environmental contamination,
and remediation are viewed. Risk communication also occurs within
this context, adding further detail to an already complex social
arena. The emotions, experiences, education, and risk perception
of the individual interact with the values of the community
and decision makers as the risks of low dose radiation—both
known and unknown—are publicly addressed. Understanding
these social processes is vital for improving the communication
of results from the Low Dose Radiation Research Program. The
data collection component of our work is nearly complete; we
are now conducting data analysis for several research tasks
grouped under three major headings. These are listed below along
with a very brief summary of current efforts and draft reports
in each area.
1.
Theories, Frameworks, and Concepts.
1.1
Nuclear Stigma. “Notes on the social history of radiation.”
Historical conditions provide critical insights into current
public attitudes about radiation and help clarify the existing
and potential role for radiation risk communication.
Flynn,
J. (forthcoming, Summer 2002). Nuclear stigma: Some notes on
the social history of radiation. In Pidgeon, N., Kasperson,
R., and Slovic, P. (Eds.), The social amplification of risk.
London: Cambridge University Press.
1.2.
Social Geography of Risk Communication
A second major context for risk communication is the social
geography of risk communication, a conceptual framework in which
to address the social context for risk messages, evaluations,
and outcomes.
Flynn,
J., and MacGregor, D.G. (2002). The social geography of risk
(Draft report). Eugene, OR: Decision Research.
2. Experimental Studies in Social Psychology.
2.1.
Perceptions of Radiation
Decision Research has conducted experiments in which people
evaluate and rate various natural and technological radiation
sources, as well as three scientific models for assessing radiation
risk.
MacGregor,
D.G., Flynn, J., Slovic, P., and Mertz, C.K. (2002) Perception
of radiation exposure, Part I: Perception of risk and judgments
of harm (Draft report). Eugene, OR: Decision Research.
MacGregor,
D.G., Flynn, J., Mertz, C. K., and Slovic, P. (2002) Perception
of radiation exposure, Part II: Communicating about radiation
exposure and health effects (Draft report). Eugene, OR: Decision
Research.
MacGregor,
D.G. and Flynn, J. (2002) Public perception of nuclear materials
in space research (Draft report). Eugene, OR: Decision Research.
2.2.
Emotional & Affective Responses to Radiation
In this survey experiment we have utilized a series of intuitive
scales to show how emotion, worldviews, and situational appraisals
guide people to differential evaluations about specific radioactive
exposure conditions.
Peters,
E., Flynn, J., and Slovic, P. (2002) An emotion-based model
of stigma susceptibility: Worldviews, affective reactivity,
and appraisals in the generation of stigma (Draft report). Eugene,
OR: Decision Research.
2.3.
Effects of Science Education on Radiation Decisions
We are currently administering an experiment using a tutorial
on radiation science to measure the effect of this information
on risk judgments and decisions in response to a variety of
radiation exposures.
MacGregor,
D.G. (2002). Ionizing radiation: A tutorial on sources and exposures
(Draft report). Eugene, OR: Decision Research.
MacGregor,
D.G., Mertz, C.K. (2002) Communicating models of radiation health
effects: The role of knowledge and perceptions (Draft report).
Eugene, OR: Decision Research.
3. Community and Small Group Studies
3.1
Small Group Studies
We have completed some exploratory small group studies to examine
the potential for negotiated decisions about radiation exposure
risks.
Gregory,
R. and Arvai, J. (2002). A decision-focused approach to understanding
DOE cleanup priorities (Draft report). Eugene, OR: Decision
Research.
3.2
Community Case Studies
Three
case studies are designed to examine how social groups, communities,
and information flows influence responses to radiation issues
at the community level near DOE facilities at Rocky Flats, CO,
Fernald, OH, and Brookhaven, NY.
Satterfield,
T. and Levin, J. (2002) Public science, fugitive values, and
the problem of
tradeoffs at Rocky Flats (Draft report). Eugene, OR: Decision
Research.
Tuler,
S. (2002) Low dose risk perception and communication: A case
study of on-site
remediation and off-site community health studies at the Fernald
nuclear weapons
site. (Draft report). Leverett, MA: Social and Environmental
Research Institute.
Webler,
T. (2002). Low dose risk perception and communication: A case
study of the tritium controversy at Brookhaven National Laboratory
(Draft report). Leverett, MA: Social and Environmental Research
Institute.
.
Mertz, C.K., Flynn, J., MacGregor, D., Satterfield, T., Johnson,
S., Tuler, S., and Webler, T. A descriptive report on community
surveys for Rocky Flats, CO, and Fernald, OH (Draft report).
Eugene, OR: Decision Research.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: DNA Damage Clusters
in Human Cells: Endogenous Levels, Induction by Radiation, and
Repair.
Authors:
Betsy M. Sutherland1, Paula V. Bennett1, Nela Cintron-Torres1,
Alexandros Georgikilas1, Peter Guida1, Megumi Hada1, Denise
Monteleone1, Sunirmal Paul1, Helga Schenk1, Joon-Myong Song1,2,
John Trunk1 and John Sutherland1,3.
Institutions:
1Biology Department, Brookhaven National Laboratory,
2 Current address, Life Science Division,
3
Physics Department, East Carolina University.
Bistranded DNA damage clusters-two or more oxidized bases, abasic
sites or strand breaks on opposing DNA strands within a few
helical turns—are hypothesized to be critical biological
lesions that may be difficult for cells to repair. Since an
important issue is the endogenous levels of such damages, we
have asked whether repair-proficient human cells accumulate
clusters under conditions of low stress (high antioxidant, specific
vitamin and mineral supplements) or high stress (low antioxidant
and supplement levels). We measured Nth-“OxyPyrimidine”
clusters (recognized by E. coli Nth protein), Fpg-“OxyPurine
Clusters (recognized by E. coli Fpg protein), Nfo-“Abasic”
clusters, (recognized by E. coli Nfo protein) as well
as double strand breaks. Under both growth conditions, the level
of endogenous clusters in 28SC monocytes, (repair-proficient
human cells) is virtually zero.
Clusters
are induced by low doses of low linear energy transfer radiation
in human cells; the dose response functions for the induction
of are linear over the dose range 0-50 cGy. The ratios of such
clusters are 1 double strand break to 1 OxyPurine cluster to
0.9 OxyPyrimidine cluster to 0.75 Abasic cluster. Thus double
strand breaks apparently comprise less than 30% of the complex
DNA damages. The proportions of clustered damages in human cells
are more similar to those we found previously for T7 DNA irradiated
in radioquenching Tris buffer (1 DSB: 1.4 OxyPurine cluster
to 0.75 OxyPyrimidine cluster to 0.4 Abasic cluster.) than to
the same DNA irradiated in non-radioquenching phosphate buffer
(1 DSB: 2 OxyPurine clusters: 0.6 OxyPyrimidine cluster: 1.5
Abasic cluster). Both the absolute levels of complex damages
and the relative proportions of the different cluster types
depend on the environment of the DNA. These data suggest that
levels of small (oxidants, anti-oxidants, quenchers, etc) and
large (structural proteins, enzymes, other nucleic acids, membrane
components) molecules in the DNA milieu can affect the levels
and types of complex damages in cellular DNA.
Since
abasic clusters are produced directly by radiation, and could
be intermediates in repair of other clustered damages, we determined
their repair—as well as processing of double strand breaks—in
repair-competent human cells. DSBs induced by low doses of X-rays
are rapidly and effectively removed, within a few minutes after
irradiation. Subsequently, de novo DSBs are generated, but contrary
to expectations, at levels apparently corresponding to only
about 10-12 % of the (non-DSB) clustered damages that potentially
could be converted to DSBs. Radiation-induced Abasic clusters,
however, are removed only slowly; further, de novo abasic clusters
appear, presumably as repair-intermediates in the processing
of other complex damages. These data suggest that human cells
may avoid production of double strand breaks in repair of clustered
damages, and instead produce repair-intermediate clusters. We
anticipate that, in addition to de novo abasic clusters, human
cells may also generate repair-intermediates oxidized base clusters
in repair of complex damages.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Markers of the Low-Dose Radiation Response.
Author:
William S. Dynan.
One of the major challenges in the field of radiation biology
is to correlate the results of biochemical studies with the
process of DNA repair as it occurs in the living cell. The overall
goal of our project is to develop better methods for visualizing
DNA double-strand break (DSB) repair complexes in situ,
in irradiated cells. Technologies are particularly needed to
study repair complexes induced by low doses of radiation, where
only one or a few breaks may be present.
Our
approach for in situ detection of DSBs involves the
development of recombinant single chain antibodies, or scFvs,
that recognize active repair complexes and other markers that
localize at DSB sites. ScFvs consist of antibody heavy and light
chain genes fused through a flexible linker. Unlike conventional
antibodies, they are extremely versatile. They can be engineered
with precise specificities, enabling them to recognize proteins
in an active conformation or phosphorylation state.
Even
with the use of scFv technology, identification of repair complexes
at low doses is challenging because of the inherent difficulty
of distinguishing a low-level signal from various forms of background
noise. Several strategies may help overcome this problem. One
is to take advantage of biological amplification, where a single
break engenders formation of multiple, spatially localized molecular
markers. The best-characterized example of biological amplification
is the generation of megabase domains of modified chromatin,
on either side of a DSB, containing the gamma-H2AX phosphorylated
histone variant. A second strategy is to develop mechanism-based
inhibitors, capable of arresting DSB repair midway through the
process, thus prolonging the lifetime of ephemeral repair complexes.
We hypothesize that these strategies will afford inherent synergy.
That is, the presence of arrested repair complexes should lead
to further biological amplification of DSB markers at the site
of the break.
Here
we describe progress toward development of a mechanism-based
inhibitor that can be used to prolong the lifetime of DSB
repair complexes. The inhibitor consists of a single chain
antibody, scFv 18-2, that was derived from a well-characterized
monoclonal antibody parent. The primary sequence of scFv 18-2
has been determined and a three-dimensional model has been
constructed, providing a framework for further optimization
of its characteristics. The recombinant antibody is active
in ELISA and immunoblot assays, and the binding affinity for
its target epitope has been determined using surface plasmon
resonance. ScFv 18-2 is a very effective inhibitor of DSB
repair in a cell-free system. Importantly, epitope mapping
shows that scFv 18-2 interacts with a short sequence near
residue 2000 of the 4127 residue DNA-PKcs polypeptide. This
is in sharp contrast to the few known pharmacological inhibitors
of DNA-PKcs, which target the kinase domain near the C-terminus.
The kinase domain is shared among members of a multigene family,
whereas the scFv 18-2 epitope is unique to DNA-PKcs. This
characteristic will allow scFv 18-2 to be used to selectively
inhibit DSB repair without perturbing signaling processes
mediated by other kinase family members. ScFv 18-2 will be
useful for in situ studies of repair complexes and for other
applications.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: The role of somatic intrachromosomal
recombination in response to low-dose X-radiation induced
damage.
Authors:
Pamela J. Sykes.
Institutions:
Department of Haematology and Genetic Pathology, Flinders
University and Medical Centre.
The
pKZ1 transgenic recombination mutagenesis model enables sensitive
detection of DNA inversion events via a somatic intrachromosomal
recombination (SICR) mechanism. The transgenic construct consists
of an E.coli b-galactosidase (lacZ) gene in inverse
orientation with respect to a promoter-enhancer complex. When
an SICR inversion event occurs within the transgenic construct,
the lacZ gene may be expressed. The b-galactosidase (b-gal)
protein product is subsequently detected using histochemical
staining in tissue sections with the chromogenic substrate
X-gal, which stains blue. Inversions and deletions in the
transgene can also be detected by polymerase chain reaction.
We have previously demonstrated changes in the levels of inversions
in spleen in pKZ1 animals in response to a number of Ames
negative and Ames positive DNA damaging agents. For example,
we have shown induction of inversions in response to cyclophosphamide
at doses 4 orders of magnitude lower than other mouse models
have been able to identify point mutations.
Our
preliminary results with X-ray exposure again indicate the
sensitivity of the pKZ1 recombination mutagenesis model. We
have shown a 4.2-fold induction of inversions in pKZ1 spleen
3 days after a 200 rad whole body dose of X-rays and a 2-fold
induction of inversion events in pKZ1 spleen after a single
exposure of 10 rad X-rays. Ten rad is 10-fold lower than doses
which have previously been shown to induce mutations in other
mouse mutation models. We have also developed a pKZ1 hybridoma
cell line which responds to X-irradiation in a similar manner
to that observed in spleen cells in the whole animal. We observed
a 3 - and 1.8 - fold induction of inversions in response to
200 rad and 10 rad X-rays in the cell line, respectively.
This suggests that the cell line will provide a useful model
for the study of mechanism of response to low dose X-rays.
We will now investigate doses lower than 10 rad in both the
pKZ1 mice and the pKZ1 cell line. We are also presently developing
a quantitative real-time PCR to detect the inversion events.
This will provide a more rapid method for quantifying the
inversions compared with histochemistry, and will also enable
larger numbers of cells to be screened efficiently.
We
are in the process of establishing a colony of pKZ1 mice which
are heterozygous or homozygous knock-outs for the ataxia telangiectasia
(atm) gene. Atm knock-out mice are susceptible to DNA damage
from radiation. We will compare the sensitivity of the atm
heterozygotes and homozygotes to low dose X-rays compared
with normal mice. Other aspects of the project that we are
about to commence are investigation of adaptive response and
long-term genomic instability after exposure to low dose radiation.
This
project is supported by the Low Dose Radiation Research Program,
Biological and Environmental Research (BER), U.S. Department
of Energy, Grant No. DE-FG02-01ER63227.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title:
The Induction of Truly Simple Exchanges Is Not Independent
of Dose Rate.
Authors:
B.D. Loucas1 , S.M. Bailey2, E.H. Goodwin3 and M.N. Cornforth1.
Institutions:
1Dept. Radiation Oncology, Univ. Texas Medical Branch,
2Dept. Radiol. Health Sciences, Colorado State Univ.,
3Biosciences Division, Los Alamos National Laboratory.
For many years it was assumed that virtually all radiation-induced
exchange aberrations were “simple”, arising through
the pairwise rejoining of two breaks. Subsequent whole chromosome
painting studies led to the realization that exchanges are
frequently complex, involving the interaction of three (or
more) damaged breaks distributed among two (or more) chromosomes.
Because such studies typically involve painting only a small
number of select chromosomes, ambiguities arise in the resulting
staining patterns that confound attempts to estimate the frequency
and extent of complex rearrangements. Many complex xchanges
produce pseudosimple staining patterns, meaning they only
appear to
be simple. And while other exchanges can often be identified
as being complex by their staining patterns, the number of
chromosomes and breakpoints which can be deduced to have participated
in the exchange often severely underestimates that which has
actually occurred. These ambiguities are largely overcome
through the use of modern combinatorial painting techniques,
such as mFISH or SKY, that allow the identification of each
homologous chromosome pair in the human karyotype.
An
astonishing prediction that arose from the analysis of earlier
whole chromosome painting data is that the characteristic
curvature in the low LET dose response for chromosome aberrations
derives principally (if not solely) from complex aberrations,
leaving the dose response for simple exchanges with an apparently
linear shape. This prediction was later experimentally verified
by our own mFISH studies on human lymphocytes and fibroblasts.
These results have been viewed as a challenge to the usual
cytogenetic viewpoint that exchanges involve the interaction
of two (or more) damaged sites, via a molecular process employing
nonhomologous endjoining. Thus, such interaction is fundamentally
two-hit in nature. That the dose response for simple exchanges
has a linear shape has been interpreted by some investigators
to support the alternative notion that exchanges occur when
a single radiation-induced chromosome break enters into an
exchange with an undamaged chromosome via a one-hit process,
a notion seemingly compatible with repair processes utilizing
homologous/homeologous recombination.
The
issue is of primary relevance to low dose effects, because
if a one-hit mechanism is really responsible for the formation
of simple exchanges, then the linearity in question defines
a dose response that, by definition, can be extrapolated with
confidence to arbitrarily low (e.g., sub-rad) doses. Significantly,
a one-hit mechanism also predicts that the results of such
an extrapolation would be the same, irrespective of radiation
intensity. In other words, the dose response for simple exchanges
should be identical, regardless of the rate at which low LET
radiation is delivered. To test this prediction we irradiated
noncycling G0 human fibroblasts with 137Cs g–rays under
conditions of limiting low dose rate (LLDR). [LLDR is defined
here as a dose rate for which further reduction in dose rate
does not lead to additional reduction in the frequency of
chromosome aberrations]. Results were compared to those derived
from cells receiving comparable doses given at high (acute)
dose rates.
To
address the issue definitively, we employed mFISH, which allowed
us to distinguish unequivocally the truly simple exchanges
from pseudosimple exchanges. Our previously reported preliminary
results showed that the acute (high dose-rate) dose response
(slope) for true simple exchanges was significantly steeper
than that obtained under LLDR. We now extend these results
to include the analysis of additional cells, including those
from acutely irradiated cultures that were given the benefit
of full postirradiation recovery prior to release from density
inhibition (i.e., PLDR). This was deemed necessary in order
to make a more meaningful comparison to cells exposed at LLDR,
as the vast majority of damage under these conditions is subject
to full PLDR. We can now state with considerable confidence
that the dose response for simple exchanges from acute radiation
exposures is fully five-fold higher than that produced under
conditions of LLDR. Consequently, while the acute dose response
for simple exchanges is, in fact, largely linear in shape,
it is unlikely that this linearity derives from a one-hit
process. Instead, we argue that the process of complex exchange
formation competes for broken chromosome ends that might otherwise
become involved in the formation of simple exchanges. Competition
for reactive breaks causes warpage in the shape of dose response
for simple exchanges which, over a limited range of doses,
gives the appearance of linearity with dose.
Thus,
the data do not support the contention that the apparent linearity
observed in the acute dose response for simple exchanges derives
from a one-hit interaction process. On the other hand, the
data are generally consistent with the predictions of the
classical two-hit model. An interesting observation less easily
explained by either model relates to the appearance of complex
aberrations under LLDR. We now estimate that roughly ten percent
of all exchange breakpoints derive from complexes under LLDR,
a value that should hold for situations of extremely low doses
delivered acutely. For both models, the contribution of complexes
to overall cytogenetic damage would be expected to (and does)
fall with decreasing dose and decreasing dose rate. But since
aberrations produced under LLDR are assumed to arise exclusively
by single track action for both models, it is surprising that
any complex aberrations would be produced at all.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Radiation Sensitivity and Cancer Susceptibility.
Authors:
Jeffrey L. Schwartz1, H. Joachim Deeg2, and Wendy Leisenring2.
Institutions:
1University of Washington and 2Fred Hutchinson Cancer Research
Center.
The goal of this study is to identify genetic factors that
affect individual susceptibility to low dose radiation. Our
working hypothesis is that individual variations in radiosensitivity
are inherited traits that define risks for radiation-induced
cancer. Our long-term goal is to identify radiosensitive and
cancer susceptible individuals from an exposed population,
then characterize susceptibility factors and identify the
responsible genetic elements. In this study we focused on
developing risk estimates for second cancer development in
patients receiving total body irradiation (TBI) as part of
a conditioning regimen for bone marrow transplantation. Between
1969 and 2000 there were 2679 aplastic anemia, myelodysplastic
syndrome, and chronic myelogenous leukemia patients treated
with allogeneic transplants at our institution. We limited
our analysis to 1568 patients who survived at least one year
after transplant to try and insure time for the development
of a second malignancy. Amongst these 1568 patients, 102 developed
a second malignancy, of which 87 were solid tumors. Most of
the solid tumors were skin carcinomas, but there are a growing
number of breast cancers developing with time (significant
with >10 years of follow-up). Risk increased with increasing
time of follow-up. The longer a patient survives exposure,
the greater the likelihood of developing a solid tumor. The
average fractionated radiation dose was 12 Gy yielding a rate
of induction of about 0.5%/Sv. This is between 2.5- to 5-times
the rate estimated in BEIR V. Patients who received radiation
had more than 2.5 times the hazard of developing a solid tumor
than did patients receiving chemotherapy for bone marrow conditioning.
In contrast to BEIR V, our study suggests that older age at
exposure is a significant risk factor. The risk of developing
a second malignancy in patients who are over 40 years of age
is 4.3 times that of patients under 18 years old.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title:
Mechanisms of Enhanced Cell killing at Llow Doses: Implications
for Radiation Risk.
Authors:
P. J. Johnston and G. D. Wilson.
Institutions:
Gray Cancer Institute.
We have found that small acute (<0.5Gy) or very low dose
rate irradiation is more lethal per unit dose than previously
predicted. At higher doses or dose rates, cells are increasingly
resistant per unit dose. These phenomena have been termed
low-dose hyper-radiosensitivity (HRS) and increased radiation
resistance (IRR). We are currently examining the mechanisms
of both HRS and IRR, in particular, the signaling pathways
involved.
It
has been reported that exposure to 3-aminobenzamide (3-AB),
an inhibitor of poly (ADP-ribosyl) polymerase (PARP) modifies
HRS/IRR by preventing the expression of IRR. We have extended
these observations by examining the effect of activators and
inhibitors of PARP. Doses of radiation that induce radiation
resistance also abrogate the toxic effects of 3-AB. Non-toxic
doses of the inhibitor 8-hydroxy-2-methylquinazolin-4-one
(NU1025) have shown the inhibition of IRR in T98G cells (HRS/IRR
positive) while no significant modification of clonogenic
survival was evident in U373 cells (HRS/IRR negative).
HRS/IRR
is most marked in the G2 phase of the cell cycle. Asynchronous
cells previously identified as failing to exhibit HRS also
exhibit marked low dose hyper-radiosensitivity when synchronized
in G2 by cell sorting. Treatment of cells with on-toxic doses
of caffeine enhances low dose hypersensitivity but has lesser
effects at higher doses of ionizing radiation indicating a
possible role for the caffeine sensitive cell cycle checkpoints
in IRR.
Direct
and circumstantial evidence has indicated that DNA double
strand break repair via non-homologous end joining (NHEJ)
is probably the process most closely connected with IRR. We
have therefore examined the role of DNA dependent protein
kinase in HRS/IRR. Cell lines deficient for the key DSB repair
enzyme DNA dependent protein kinase (DNA-PK) fail to exhibit
IRR. Similarly, non-toxic concentration of wortmannin, an
inhibitor of DNA-PK, radiosensitized both T89G and U373 cells
abrogating the IRR type response. Consistent with published
reports, preliminary Western blotting experiments indicate
that the levels of DNA-PKcs, Ku70 and Ku80 are unchanged in
response to low doses of ionizing radiation in the dose range
(0.05-0.8 Gy) and time course (0.5-2 hours) of IRR. It has
been reported that a significant correlation exists between
changes in DNA-PK activity in response to irradiation and
the extent of IRR. We have been unable to confirm these findings.
No significant changes in DNA-PK activity were observed in
HRS/IRR positive/negative cells over the dose range and time
course of IRR. It appears that current kinase assays are not
sufficiently sensitive to detect very small (<20%) changes
in DNA-PK activity with reproducibility.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title:
Dysfunctional Telomeres, Radiation-Induced Instability and
Tumorigenesis.
Authors:
Susan M. Bailey1, Edwin H. Goodwin2, Michael N. Cornforth3
and Robert L. Ullrich1.
Institutions:
1Dept. of Radiological Health Sciences, Colorado State University,
2Bioscience Division, Los Alamos National Laboratory,
3University of Texas Medical Branch.
Telomeres are highly specialized nucleoprotein structures
that stabilize and protect the ends of linear chromosomes.
As such, telomeres play an essential role in preserving the
integrity of eukaryotic genomes, a function they normally
perform very well. However, when telomere dysfunction does
occur, the consequences can be severe, including cellular
senescence and the formation of chromosomal rearrangements
likely to be associated with carcinogenesis. Previously, we
demonstrated that effective end-capping of mammalian telomeres
has a seemingly paradoxical requirement for proteins more
commonly associated with DNA double-strand break (DSB) repair.
Ku70, Ku80, and DNA-PKcs (the catalytic subunit of DNA-dependent
protein kinase) all participate in DSB repair through non-homologous
end-joining (NHEJ). Somewhat surprisingly, mutations in any
of these genes cause spontaneous chromosomal end-to-end fusions
that maintain large blocks of telomeric sequence at the points
of fusion. The fusions¾ which contribute significantly
to the background level of chromosomal aberrations [Bailey
et al., PNAS 96 (1999), 14899]¾ are not a consequence
of telomere shortening. We have also shown that nascent telomeres
produced via leading-strand DNA synthesis are especially susceptible
to these end-to-end fusions, suggesting a crucial difference
in postreplicative processing of telomeres that is linked
to their mode of replication [Bailey et al., Science 293 (2001),
2462]. Here we report that impaired end-capping in DNA-PKcs-deficient
genetic backgrounds not only allows dysfunctional telomeres
to join to each other, but also to broken chromosome ends
created by radiation-induced DSB. Mouse cell lines were exposed
to graded doses of gamma-rays and examined utilizing the strand-specific
fluorescence in situ hybridization technique of CO-FISH, in
order to distinguish true telomere-to-DSB events from telomere-to-telomere
fusions. Telomere-to-DSB fusion events were observed in a
dose-dependent fashion in each mutant cell line analyzed,
including BALB/c, which is both radiosensitive and susceptible
to radiogenic mammary cancer. The BALB/c phenotype has been
attributed to a variant allele of the DNA-PKcs gene, Prkd-cBALB,
which has two naturally occurring coding sequence polymorphisms
that result in reduced DNA-PKcs abundance and activity, most
markedly in mammary gland tissue [Yu et al., Cancer Research
61 (2001), 1820]. Our results demonstrate that dysfunctional
telomeres in cells with DNA-PKcs deficiency can inappropriately
fuse to DSB ends, creating novel chromosome structural rearrangements
that maintain large blocks of interstitial telomeric sequence.
Thus, beyond their established role in maintaining the lengths
of terminal sequences, telomeres have additional capping functions
that involve both chromosomal radiosensitivity and genomic
stability. Currently under investigation with respect to tumorigenesis,
are the relationships between deficiencies in radiation response
genes and the concomitant generation of dysfunctional telomeres.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Low dose hypersensitivity and bystander
responses in human and mouse
fibroblasts: A comparison of conventional and focused soft
x-rays.
Authors:
Elena V. Rusyn1, Gieseppe Schettino2, Melvyn Folkard2, Kevin
M. Prise2, Barry D. Michael2, and Kathryn D. Held1.
Institutions:
1Massachusetts General Hospital and 2Gray Cancer Institute.
It has been shown that hypersensitivity to low doses of radiation
occurs in a range of animal and human tumor cell lines. However,
little is known about the response of primary human cells.
Here, primary human fibroblasts (AGO1522) were exposed to
low doses of conventional X-rays or focused soft X-rays. The
results show that at doses of 0.2 Gy and below of conventional
X-rays hypersensitivity with respect to cell clonogenicity
was observed. Furthermore, a similar hypersensitive response
to the same doses of conventional X-rays was found when the
production of micronuclei was measured. When individual cells
were irradiated through the nucleus with a focused carbon-K
soft X-ray microprobe, cells were more radiosensitive compared
to conventional X-rays as measured by both the clonogenic
survival and micronucleus formation assays at doses greater
than 0.2 Gy. However, no hypersensitivity to low doses of
focused soft X-rays was observed. To test whether induction
of intracellular reactive oxygen species and oxidant-antioxidant
balance are involved in the mechanism of hypersensitivity
to conventional X-rays dimethyl sulfoxide, a hydroxyl radical
scavenger, and buthionine sulfoximine, a suppressor of intracellular
glutathione production were used. Dimethyl sulfoxide had no
protective effect on the hypersensitive response of cells
to conventional X-ray irradiation. However, pretreatment of
cells with buthionine sulfoximine before irradiation had a
radiosensitizing effect with respect to cell survival at all
doses, and the non-linearity of the dose-effect relationship
at 0.2 Gy and below was not observed.
In
hamster V79 cells, we have been studying the relationship
between low-dose hypersensitivity and bystander responses
using focused soft X-rays targeted through the nucleus. At
low doses (< 0.2 Gy), we have observed the same degree
of cell killing, regardless of whether every cell within a
population or only a single cell within a population is targeted.
This suggests that bystander responses predominate at low
doses and that every cell can produce a bystander signal.
Further studies are determining the role of cell cycle position
on the degree of bystander response observed, by using a computerized
imaging system to classify cells according to their cell cycle
position at the time of irradiation. Preliminary data suggest
no significant relationship with the cycle phase of the cells
that respond to the bystander signal.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Effects of Low Dose Ionizing Radiation
on Gene Expression in Human Skin.
Authors:
Zelanna Goldberg(1), Chad W. Schwietert(1), Robin L. Stern
(1), Michelle Arnold (2), Christine L. Hartmann Siantar (2),
Robert Cary (3), Marie-Anne Descalle (2), and Bruce E. Lehnert
(3).
Institutions:
1 University of California, Davis, Dept. of Radiation Oncology,
2 Laurence Livermore National Laboratory,
3 Los Alamos National Laboratory.
Significant biological effects can occur in animals, animal
cells, immortalized human cell lines, and primary human cells
after exposure to doses of ionizing radiation (IR) in the
<1-10 cGy region. How these and other observations mimic
or even pertain to the actual condition, especially in humans
is unclear, though such knowledge is ultimately required for
reducing the uncertainty of assessing human risks due to low
dose IR (LDIR) exposures. Thus, human translational data must
be obtained with which to correlate in vitro experimental
findings and evaluate their “real-life” applicability.
Our project uses human skin, irradiated In vivo during
therapeutic radiation as a model system. Preliminary studies
have focused on verifying the accuracy of the dosimetry in
the low dose, out of field areas, optimizing RNA and protein
extraction from the samples, assessing RNA amplification strategies
and performing microarray analyses to ensure the robustness
of the physics and biology components of the project prior
to obtaining patient samples.
We
combined measurements and PEREGRINE 3D Monte Carlo simulations
to establish an overall 10-15% uncertainty predictive capability
for the 18 MV clinical radiation beam used in these experiments.
Based on our findings, we have altered our paradigm for sample
collection: we will collect samples at the exit surface of
the patient in order to optimize dosimetric accuracy and minimize
dose gradient through the sample. Combining real-time measurement
and exit-surface collection we anticipate overall dosimetric
uncertainties of 5%.
Preliminary
biologic studies have focused on obtaining global gene expression
data from small volume human skin samples. Samples have been
obtained from resected tissue from elective surgical procedures.
3 mm diameter core skin biopsies have been performed and samples
from different areas of the body have been compared within
a given person to examine homogeneity of the skin site sampled.
Tissue samples are incubated up to 24 hours to assess stability
of message, or they are subjected to immediate ex vivo IR
at 1, 10 or 100 cGy and then incubated for equivalent times.
RNA is extracted, processed, and hybridized to cDNA microarrays
containing over 12,500 unique sequence validated human cDNA
clones to assess gene expression changes in the samples. Expression
profiles generated from amplified and unamplified RNA are
being compared to confirm the fidelity of amplification schemes
that are required for samples containing limited RNA.
Preliminary
gene expression microarray hybridization data have suggested
that as many as 116 genes have altered expression of at least
a 2-fold extent following 1 cGy exposure ex vivo. These include
genes that have been shown to radioresponsive in pure in vitro
cell systems.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Low-Dose Radiation Induced Changes
in Gene Expression in Mouse Brain and Testis.
Authors:
E. Yin1, M.A. Coleman1, L.E. Peterson2 and A.J. Wyrobek1,
Institutions:
1. Biology & Biotechnology Research Program, Lawrence
Livermore National Laboratory. 2. Department of Medicine,
Baylor College of Medicine.
Radiation is known to induce genetic instability, which in
turn may result in the formation of aberrant cells that progress
to tissue pathology and tumorigenicity. It has been hypothesized
that this process occurs through alterations in DNA replication
and repair pathways. It is also known that various tissues
within living organisms have different characteristic responses
to environmental challenges. The brain is a mitotically quiescent
tissue which is relatively radio-resistant, capable of withstanding
up to 50Gy before lethality. Testis, on the other hand, is
relatively radio-sensitive, and shows considerable germ-cell
toxicity at doses below 2Gy. We hypothesize that changes in
gene expression within these two tissues after radiation exposure
will be related to their degree of radio-sensitivity, and
that the genes which show altered expression within the first
few hours of low-dose irradiation are pivotal in determining
the fate of irradiated cells. We have examined the gene expression
profile of both brain and testis in B6C3F1 mice irradiated
with either 0.1Gy or 2Gy at both early (30 min.) and later
(4 hr.) time points. Within each tissue, there are numerous
genes that showed differential expression after low-dose exposure
as determined by statistical analysis. Results from clustering
analysis (CLUSFAVOR) indicate that there are large groups
of genes that show both similar and significant differences
in response as well as tissue specificity. [This work was
performed under the auspices of the U. S. Department of Energy
by the University of California, Lawrence Livermore National
Laboratory under Contract No. W-7405-Eng-48.]
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Radiation response in normal HSF (human
skin fibroblasts): cDNA microarray analysis.
Authors:
Elina Golder-Novoselsky, Liang-Hao Ding, Fanqin Chen, David
J. Chen.
Institutions:
Lawrence Berkeley National Laboratory, Life Sciences Division
The advancement of microarray technology is having a major
impact on our understanding of a variety of biological processes.
Radiation-induced damage and repair is still one of the processes
that is poorly understood. Though there have been a few studies
addressing radiation-induced gene expression in tumor cell
lines, there is no comprehensive set of data available for
primary cell cultures. Using cDNA microarray technology, we
set out to investigate the global transcriptional effects
of LLIR, using HSF, primary human skin fibroblasts. These
cDNA microarrays, designed and printed in our facility, contain
8000 known sequence-confirmed genes.
Using
rigorous computational methods, we characterized the dose-dependent
radiation- induced gene expression of HSF-42, a primary cell
culture. Our preliminary results demonstrate that there are
discrete groups of low (0.02 Gy), intermediate (1 Gy) and
high (4 Gy) dose-regulated genes. Using these doses, as well
as a time course (1, 2, 4, 24 hrs), we show that there are
detectable responses with doses of X-rays as low as 0.02 Gy
and as early as 1-2 hrs post-irradiation. These include a
variety of targets involved in a multitude of cellular responses,
such as apoptosis, adhesion, cell cycle, and DNA repair. Some
examples of down-regulated genes are integrins, metalloproteinases,
caspases (1 and 8), topoisomerase, VEGF, BMP2, interleukins,
collagens, and TGF-beta. The up-regulated group is represented
by diverse genes like integrins, BCL2, caspase 9, CHK1, Tre-2
oncogene, RAD51-interacting protein, DNA ligase III, p21,
and RAP1A. In addition, we have observed a radiation dose-response
among many genes—for example, IGBP1 and MMP3 are down-regulated
by nearly 7-fold with the dose increase from 0.02 Gy to 4
Gy, while SPARC, NID2, BLCAP, RLF, p21 are 2- to 5-fold up-regulated
with increased dose of radiation.
In
summary: our preliminary results demonstrate that in HSF primary
cells, expression of a large number of genes is not only regulated
by LLIR, it is also regulated in a dose and time-dependent
manner.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Genome-scale Modeling of Low-Dose
Irradiation Responses Using Microarray Based Gene Networks.
Authors:
Matthew A. Coleman1, Leif Peterson2, Terence Critchlow1, and
Andrew J. Wyrobek1.
Institutions:
1. Biology & Biotechnology Research Program, Lawrence
Livermore National. Laboratory. 2. Department of Medicine,
Baylor College of Medicine.
Participating Consortium members: Tom Slezak
(LLNL), Dave Nelson (LLNL), Bertram Ludaescher (LBL), Amarnath
Gupta (LBL), Ilkay Altintas (LBL), Tom Potok (ORNL), Mladen
Vouk (NCSU), Calton Pu (Georgia Tech.), Ling Liu (Georgia
Tech.), David Buttler (Georgia Tech.), Dan Rocco (Georgia
Tech.), Henrique Paques (Georgia Tech.), Wei Han (Georgia
Tech.).
Cells
and tissues with similar radiation response phenotypes are
predicted to have common ionizing radiation (IR)-induced gene
expression profiles that are controlled by shared groups of
regulatory elements (also known as synergistic gene groups).
Our overall objective is to utilize genome-scale expression
microarray data in conjunction with DNA sequence/pattern databases
available on the Web, to build a computer-based gene-network
model for identifying, grouping and predicting regulatory
elements that control differential aspects of the early cellular
responses to IR. This research project is defined by: (1)
Grouping genes identified by microarray experiments into IR-responsive
clusters based on their relative-transcript abundance and
their differential IR radiation responses at low (10cGy) and
high doses, (2) Identifying regulatory elements (and their
locations relative to the open reading frame) that distinguish
among separate IR responsive gene clusters and (3) Comparing
IR responsive gene clusters identified “in silico”
to those IR responsive genes identified on microarrays in
many different laboratories. To accomplish this we have brought
together a diverse group of investigators to build a gene
pathway model of the cellular controls of radiation response.
This project takes advantage of stand alone software (CLUSFAVOR,
http://mbcr.bcm.tmc.edu/genepi/) to identify clusters and
groups of interesting IR responsive genes that are then used
to search relational database systems (http://www.llnl.gov/CASC/datafoundry/index.html,
SDM http://sdm.lbl.gov/sdmcenter/)
to parse information from genomic databases such as
GenBank, KEGG and UniGene. Obtained sequence information is
then used for promoter analysis (ModelInspector, http://genomatix.gsf.de/)
to identify synergistic gene groups that share conserved regulatory
elements. This database is being designed to allow data analysis
across multiple platforms (i.e., cDNA as well as oligo-array
data). In the future, experimental low-dose radiation array
data sets from microarray experiments will be compared to
the resulting database of synergistic groups and their regulatory
elements to assess the ability and accuracy of our ability
to predicted the same IR responsive genes and pathways. The
identification and characterization of regulatory element
profiles of IR-responsive genes will provide valuable understanding
of the genetic mechanisms of IR-response and should provide
powerful biological indicators of genetic susceptibilities
for tissue and genetic damage. The resulting model is being
developed as a foundation for a unique predictor of new genes
and for testing new hypotheses related to exposure of IR based
on coordinate gene/pathway interactions.
[This work was performed under the auspices of the U. S. Department
of Energy by the University of California, Lawrence Livermore
National Laboratory under Contract No. W-7405-Eng-48.]
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Quantitative Analysis of Connexin
Expression in Cultured Colonies.
Authors:
B. Parvin, Q. Yang, R. L. Henshall-Powell and M.H. Barcellos
Hoff.
We are studying the effects of ionizing radiation on the signaling
between human mammary epithelial cells and the extracellular
microenvironment. To do so we use an assay based on the ability
of the cells to organize into three-dimensional acini when
embedded into an extracellular matrix. Although tumorigenic
and non-tumorigenic mammary epithelial cells are nearly indistinguishable
when cultured as monolayers, their biological character readily
diverge when tissue-specific morphogenesis is analyzed. Non-malignant
human mammary epithelial cells (HMEC) cultured within a reconstituted
basement membrane organize into acinar-like structures with
polarity; in contrast, breast cancer cells form disorganized
aggregates similar to tumors In vivo. These are studied using
immunofluorescence and confocal microscopy, which permits
the reconstruction of 3-dimensional organization.
Automatic
detection of cell structures and localization of protein expression
from volumetric dataset is an important step in large scale
analysis of cultured colonies and their intercellular interactions.
Detection of an individual nucleus reveals morphological features
like size and shape, can be used to map the multicellular
organization of each colony, and enables localization of intercellular
signaling components as a function of treatment. The focus
of this initial study is to determine the frequency of gap
junction protein complexes. Connexins are a family of proteins
associated with gap junctions that modulate the transfer of
molecules between cells. Connexins-43 and -32 localized as
distinct aggregates between cells of HMEC acini.
We
found that automated analysis and counting of connexin aggreates
was hampered by abundant speckle noise, which has signature
similar to connexin in the volumetric dataset. The detection
of individual nuclei provides the necessary “context”
to filter speckle noise and enables automatic counting and
characterization of connexin expression. In general the nucleus
of a cell is ellipsoidal, but neighboring nuclei may overlap,
and thus making delineation a necessary component. Our experience
indicates that detection of nuclei in 2D is more complex due
to inherent lack of 3D information, however, a more efficient
techniques is needed to detect blobs in 3D. In general, analysis
of these images is complex due to the fact that nuclei of
interests (1) do not respond uniformly to the fluorescent
compounds, (2) may have many internal substructures, and (3)
overlap each other as a result of cell division. The first
step of our algorithm is extraction of elliptic features.
A multiscale representation of the image is generated and
the Hessian is computed to detect and classify each point
in the image. If the Hessian is negative (positive) definite
then the point is classified as bright (dark) elliptic feature.
This classification is then used to group similar features
using 3D connected component algorithm. Although false labeling
is unavoidable,
sufficient information can be gathered so that a higher level
technique can group partial information into a whole. This
higher level constraint is expressed in terms of convexity,
implemented as a convex hull for improved efficiency, and
applied to 3D connected components. The convex hull of a set
of points is the smallest convex set that contains the points.
In
summary, we have developed a layered computational protocol
to segment cultured colonies for simultaneous segmentation
of nuclei, characterizing their organization, and mapping
inter cellular communication. Preliminary experimental data
of connexin expression in human mammary epithelial cells surviving
low doses of ionizing radiation and the impact of chronic
exposure to the cytokine transforming growth factor $1 will
be presented.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Comparison of DNA Damage Risk from
Low-Dose Radiation and Folate
Deficiency.
Authors:
Chantal Courtemanche, Arnold C. Huang, Nicole Kerry, Bernice
Ng, and Bruce N. Ames.
Institutions:
Children’s Hospital Oakland Research Institute, Oakland,
California.
Our overall goal is to understand and quantify the real effects
of low-dose radiation by measuring direct and specific cellular
changes. However, since the background dose of radiation to
which most individuals are exposed is well below the levels
where significant biological effects, such as mutation or
tumor induction, are observed, our novel approach is to compare
the consequences of radiation to those of specific nutritional
deficiencies. By determining which of these two common stresses
at physiologically relevant doses leads to a greater amount
of DNA damage, we hope to determine whether low-dose radiation
has a significant impact on human health, relative to other
better understood risks, such as deficiencies in the vitamins
folate (about 10% of U.S. population before recent fortification),
B6 (about 10% of U.S. population), and B12 (about 14% of U.S.
elderly population), which cause incorporation of uracil in
human DNA and consequent double-strand breaks.
Our
data thus far strongly suggest that nutritionally relevant
levels of folate deficiency are likely to be a greater cellular
stress than environmentally relevant levels of radiation.
We have established human lymphocytes in cell culture and
have either treated them with irradiation or maintained them
under folate deficiency conditions. Irradiated cells and folate
deficient cells had greatly reduced growth curves above 0.5
Gy and below 24 nM folate, respectively. Cell viability, as
measured by trypan blue exclusion, was lowered inversely with
increasing amounts of radiation exposure and with increasing
folate deficiency in the cell medium. However, apoptosis was
only increased when cells were exposed at the highest radiation
dose of 5 Gy, but there was an inverse dose response in apoptosis
when cells were maintained in varying levels of folate in
the medium. We also examined the cell cycle in order to determine
in which phase most of the cells were accumulating. At the
1 Gy or higher doses, there was an increase in arrest of the
cell cycle in the G2/M phase. Folate deficient cells, on the
other hand, showed a different profile. Lymphocytes that were
cultured in physiologically relevant levels of folate deficiency
for 8 days had a cell cycle arrest in the S phase. Furthermore,
this arrest appeared to be dose-dependent on the level of
folate deficiency. When 3H-thymidine is added, the radioactivity
is rapidly incorporated into the cell, suggesting that DNA
repair is active and that uracil misincorporation is the likely
cause of the arrest. Additionally, at the low doses of 0.5
Gy or less, we did not detect any increase in DNA double-strand
breaks, even after we applied all of our DNA repair enzymes
to our assay. We have also done 4 separate experiments to
measure gene expression changes using Operon’s Stress/Aging
DNA microarray. These studies, in total, suggest that while
the cellular responses due to irradiation and folate deficiency
are somewhat different, these respective responses occur more
readily under moderate folate deficiencies than under even
moderately high doses of radiation.
In
addition to the basic comparison between radiation effects
and folate deficiency effects, we have begun work directed
at examining the interactive effects of radiation and folate
deficiency. It is likely that these results will be more relevant
to the general population because we may be able to identify
individuals who are at greater risk to the effects of low-dose
radiation or perhaps find ways, such as sufficient nutritional
supplementation, to alleviate the effects of radiation. We
have established a growth curve for human lymphocytes that
have been maintained in folate deficient medium and then irradiated
with varying doses of 0,0.25, 0.5, 1, and 5 Gy. The growth
rate is lower than the rate for either treatment alone. Cell
viability was also slightly lower for folate-deficient cells
that were subsequently irradiated than when cells were either
folate deficient only or irradiated only. Moreover, we found
a higher percentage of cells were apoptotic when they were
first folate deficient and then irradiated. For cell cycle
analysis, folate-deficient cells displayed a S phase arrest,
while folate-deficient and irradiated cells displayed an increase
in G2/M phase arrest only at the highest irradiation dose
of 5 Gy. Since the combined effect of folate deficiency and
irradiation has predominantly the same effect as folate deficiency
alone, this suggests that folate deficiency may account for
a greater amount of cellular stress than low-dose radiation.
We have also nearly completed our initial assessment of gene
expression changes using DNA microarrays. These preliminary
data seem to indicate that there is an interactive effect
between radiation and a nutritional deficiency since we observed
effects of radiation on folate deficient cells that are not
normally observed for similar radiation doses to normal cells.
For
governmental agencies, this research may provide data that
is useful for setting public health policies, and for the
general public, this research can present radiation risk in
the readily understandable terms of equivalent effects from
eating few fruits and vegetables and may assist in changing
their perception as to what levels of radiation exposure are
relevant.
_____________________________________________________________________
Office
of Biological and Environmental Research
DOE
Lowdose Radiation Program Workshop III
Abstract
_____________________________________________________________________
Title: Low dose-rate radiation effects on
gene expression.
Authors | 
