Radiation risk to low fluences of particles may be
greater than we thought
Hongning Zhou*, Masao Suzuki*†, Gerhard Randers-Pehrson*, Diane Vannais‡§, Gang Chen¶, James E. Trosko¶,
Charles A. Waldren‡§, and Tom K. Hei***
*Center for Radiological Research, College of Physicians and Surgeons, and Environmental Health Sciences, School of Public Health, Columbia University,
New York, NY 10032; ‡Department of Radiological Health Science, Colorado State University, Fort Collins, CO 80523; and ¶Department of Pediatrics
Human Development, Michigan State University, East Lansing, MI 48824
Communicated by Richard B. Setlow, Brookhaven National Laboratory, Upton, NY, October 3, 2001 (received for review August 18, 2001)
Based principally on the cancer incidence found in survivors of the
atomic bombs dropped in Hiroshima and Nagasaki, the International
Commission on Radiation Protection (ICRP) and the United
States National Council on Radiation Protection and Measurements
(NCRP) have recommended that estimates of cancer risk for low
dose exposure be extrapolated from higher doses by using a linear,
no-threshold model. This recommendation is based on the dogma
that the DNA of the nucleus is the main target for radiationinduced
genotoxicity and, as fewer cells are directly damaged, the
deleterious effects of radiation proportionally decline. In this
paper, we used a precision microbeam to target an exact fraction
(either 100% or <20%) of the cells in a confluent population and
irradiated their nuclei with exactly one particle each. We found
that the frequencies of induced mutations and chromosomal
changes in populations where some known fractions of nuclei
were hit are consistent with non-hit cells contributing significantly
to the response. In fact, irradiation of 10% of a confluent mammalian
cell population with a single particle per cell results in a
mutant yield similar to that observed when all of the cells in the
population are irradiated. This effect was significantly eliminated
in cells pretreated with a 1 mMdose of octanol, which inhibits gap
junction-mediated intercellular communication, or in cells carrying
a dominant negative connexin 43 vector. The data imply that the
relevant target for radiation mutagenesis is larger than an individual
cell and suggest a need to reconsider the validity of the
linear extrapolation in making risk estimates for low dose, high
linear-energy-transfer (LET) radiation exposure.
Radiation can cause as well as cure cancer. The risk of
developing radiation-induced cancer has traditionally been
estimated from cancer incidence among survivors of the atomic
bombs dropped in Hiroshima and Nagasaki in 1945. These data
provide the best estimate of human cancer risk over the dose
range from 20 to 250 cGy for low linear energy transfer radiation
such as X- or -rays. The cancer risk at doses below 20 cGy,
however, is uncertain and has been the subject of controversy for
decades. Both the International Commission on Radiation Protection
and the U.S. National Council on Radiation Protection
and Measurements have recommended using a linear nothreshold
extrapolation from higher doses where more accurate
risk estimates are available (1, 2). However, this approach has
drawn criticisms for being too strict on the one hand and too
conservative on the other (3). A better understanding of the
mechanisms of radiobiological effects at low doses would shed
light on the validity of the currently used model and provide a
rationale for the best estimates of risk.
Ever since X-rays were shown to induce mutation in Drosophila
and maize, it has been accepted dogma that the deleterious
effects of radiation, such as mutation and carcinogenesis, were
due mainly to direct damage to DNA. Evidence is now emerging
that extranuclear or extracellular targets are extremely important
in mediating the genotoxic effects of radiation (4–16). We
showed, for example, that irradiation of just the cellular cytoplasm
could induce mutation in the nucleus of the target cells by
a process involving oxyradicals (11). Furthermore, very low
doses of particles induced significantly higher levels of p53 in
populations of human fibroblasts than expected from the number
of cells that had actually been hit by an particle (5). The
excess in the fraction of responding cells, which received no
radiation exposure, were termed ‘‘bystanders.’’ It has been
difficult to measure the induction of mutations in populations of
mammalian cells where only a small fraction were traversed by
an exact number of particles. Here, we used a precision charged
particle beam to deliver exactly one particle through the nuclei
of a known proportion of human-hamster hybrid AL cells to
clearly ascertain the magnitude of this bystander mutagenic
effect. We found that cells irradiated with a single particle can
induce bystander mutagenic response in nonirradiated neighboring
cells, and that gap junction cell–cell communication plays
a critical role in mediating that bystander mutagenesis. Furthermore,
irradiation of 10% of a population resulted in a mutagenic
yield that was similar to when all of the cells in the population
were hit. These results are of considerable importance in reassessing
the potential genotoxic effect of low dose radiation and
suggest that the assumption of direct proportionality in radiation
risk assessment is seriously in error.
Materials and Methods
Cell and Culture Conditions. The human–hamster hybrid AL cells
containing a standard set of Chinese hamster ovary-K1 (CHO
K1) chromosomes plus a single copy of human chromosome 11
were used in this study. Chromosome 11 encodes cell surface
antigens (CD59) that render AL cells sensitive to killing by
specific monoclonal antibody E7.1 in the presence of complement.
Rabbit serum complement was from HRP (Denver, PA).
Antibody specific to the CD59 antigen was produced from
hybridoma cultures as described (17, 18). Cells were maintained
in Ham’s F-12 medium supplemented with 8% heatinactivated
FBS, 25 gml gentamycin, and 2 104Mglycine
at 37°C in a humidified 5% CO2 incubator, and passaged as
described (19, 20).
Irradiation Procedure. Approximately 500 exponentially growing
AL cells in 0.5 l volume were inoculated into each of a series of
microbeam dishes constructed by drilling a 14 inch hole in the
center of 60-mm diameter non-tissue-culture dishes as described
Abbreviations: CX10, AH1-9 cells carrying connexin 43 overexpressing construct; G2 PCC, G2
phase premature chromosome condensation.
†Present address: National Institute of Radiological Health Sciences, 4-9-1 Anagawa,
Inage-ku, Chiba, Japan.
§Present address: Radiation Effects Research Foundation (RERF), 5-2 Hijiyama Koen,
Minami-ku, Hiroshima, Japan.
**To whom reprint requests should be addressed at: Center for Radiological Research,
Vanderbilt Clinic 11-218, College of Physicians and Surgeons, Columbia University, 630
West 168th Street, New York, NY 10032. E-mail: TKH1@Columbia.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
14410–14415 PNAS December 4, 2001 vol. 98 no. 25 www.pnas.orgcgidoi10.1073pnas.251524798
(11, 13, 19). A 3.8-m-thick polypropylene film was epoxied over
the bottom of the hole, creating a miniwell that was then coated
with Cel-Tak (BD Biosciences, Bedford, MA) to enhance cell
attachment. Two days after plating, when the number of attached
cells reached an average of 2,000 per dish with 70% of
the attached cells in contact with neighboring cells, the nuclei of
attached cells were stained with a 50 nM solution of Hoechst
33342 dye for 30 min. The image analysis system then located the
centroid of each nucleus and irradiated some or all of them
randomly, one at a time, with an exact number of particles.
After irradiation, cells were maintained in the dishes for 3 days
before being removed by trypsinization and replated into culture
flasks. After culture for 4–5 days, the cells were trypsinized and
replated to measure the mutant fraction as described (19, 20).
Cytotoxicity of a Single Particle Traversal Through the Nucleus.
Conditions for assessing the clonogenic survival of cells irradiated
with a single particle have previously been described (19).
Briefly, irradiated and control cells in a series of miniwells were
trypsinized and replated into 60-mm-diameter Petri dishes for
colony formation. After incubating for 7–9 days, cultures were
fixed with formaldehyde and stained with Giemsa. The number
of colonies was counted to determine the surviving fraction as
described (11, 13, 19).
Quantification of CD59 Mutants. Determination of the mutant
fraction was carried out as described (11, 17–20). Briefly, 5 104
cells were plated into each of six 60-mm dishes in 2 ml of growth
medium, and the cultures were incubated for 2 h to allow for cell
attachment, after which 0.3% CD59 antiserum (E7.1) and 1.5%
(volvol) freshly thawed complement were added to each dish as
described (19, 20). The cultures were further incubated for 7–8
days for colony formation. At this time, the cells were fixed and
stained, and the number of CD59 mutant colonies was scored.
Controls included sets of dishes containing antiserum alone,
complement alone, or neither agent. Mutant yields in the
cultures derived from each radiation group were determined for
two consecutive weeks to ensure full expression of the mutations.
The mutant fraction at each dose was calculated as the number
of surviving colonies divided by the total number of cells plated
after correction for any nonspecific killing because of complement
alone and was expressed as the number of mutants per 105
clonogenically viable cells.
Prediction of the Mutant Yields. Predictions of the yield of mutants
where a known fraction of the cells was irradiated through the
nucleus with exactly one particle were made based on the
assumption of no bystander mutagenic effect. Mathematically,
we can predict the mutant fraction in a culture where a known
fraction of cells has been irradiated as follows.
The number of cells that were irradiated, survived, and formed
mutants is given as: FNP.E.IRMIR where F is the fraction
of cells irradiated with exactly one particle, N is the total
number of cells in the population, P.E.IR and MIR are the plating
efficiency and mutant fraction where 100% of cells have been
irradiated with exactly one particle, respectively.
The number of cells that were not irradiated, that were
attached, and that produced mutants is given as: (1 F) N
P.E.c Mc where P.E.c, and Mc are the plating efficiency and
mutant fraction of the controls, respectively.
The expected mutant fraction in population where a known
fraction (F) of cells was irradiated by a single particle is
therefore:
F N P.E.IR MIR 1 F N P.E.c Mc
F N P.E.IR 1 F N P.E.c
.
Cancel out N and divide all terms by P.E.c, then the formation
becomes
F S.F.IR MIR 1 F Mc
F S.F.IR 1 F
,
where S.F.IR is the survival fraction where 100% of the cells have
been irradiated with exactly one particle.
Treatment with Octanol. Octanol, an effective inhibitor of gap
junction communication (21), was used to investigate the role of
gap junction-mediated cell–cell communication in bystander
mutagenesis. Cells were treated with a 1-mM dose of octanol 2 h
before and maintained until 3 days after the irradiation. After
treatment, cultures were washed, trypsinized, and replated for
survival and mutagenesis as described above.
Bystander Mutagenesis in Cells Genetically Deficient in Gap Junction-
Mediated Cell–Cell Communication. To further investigate the role
of cell–cell communication in bystander mutagenesis, we transfected
AH1-9 cells (a variant of AL cells containing a hygromycin
resistant marker on chromosome 11) with either a dominant
negative connexin 43 vector or with connexin 43 expressing
vector and repeated the bystander mutagenic studies. Connexin
43 is the principal protein component of gap junctions (22).
There is good evidence that connexin of itself (assembled in a
lipid bilayer) is sufficient and necessary for the generation of gap
junction channels (23, 24). The scrape-loading assay (25) was
used to test the existence of gap junction-mediated intercellular
communication in the AH1-9-transfected cells. Briefly, confluent,
density-inhibited cells were scraped with a scalpel blade,
exposed to Lucifer yellow (0.05%) and Rhodamine dextran
solution (0.05%) for 3 min, and washed with PBS three times,
and the distance traveled by the migrating dye was determined
under a fluorescent microscope.
Detection of Chromosomal Damage. The use of premature chromosome
condensation to analyze the frequency of chromatid
break as an index of chromosomal damage has been described
(26). We chose to use a Calyculin A-inducedG2 phase premature
chromosome condensation (G2 PCC) assay to detect chromatid
damage instead of the conventional metaphase spread because
of its higher sensitivity. Immediately after irradiation, cells were
treated with Calyculin A at a final concentration of 50 nM for 30
min at 37°C. The PCC samples were prepared according to
conventional cytogenetic procedure (26, 27). Briefly, cells were
treated with 75 mM KCl for 20 min at 37°C and fixed in
methanolacetic acid (3:1). The cell suspension was dropped on
ethanol-cleaned slides, air-dried, stained with 5% Giemsa solution,
and scored under a microscope. Chromatid-type breaks,
which included chromatid breaks and acentric fragments, were
scored from a minimum of 50 G2 PCC samples per experiment.
The estimated chromatid-type breaks per cell, assuming no
interaction between irradiated and nonirradiated cells, were
similarly calculated as described above. To assess the role of gap
junctions in mediating the bystander process, lindane (40 M) or
octanol (1 mM) was added to the cultures 2 h before irradiation
as described (13).
Statistical Analysis. Data were calculated as means and standard
deviations. Comparisons of surviving fractions and induced
mutant fractions between treated groups and controls were
made by Student’s t test. A P value of 0.05 or less between groups
was considered to be significant.
Zhou et al. PNAS December 4, 2001 vol. 98 no. 25 14411
CELL BIOLOGY
Results
Bystander Mutagenesis in AL Cells Induced by a Single Particle
Through the Nucleus. Consistent with our previous finding, traversal
of the nucleus with a single particle was only slightly
cytotoxic to AL cells, resulting in a surviving fraction 0.79
0.05 (19). The yield of CD59 mutants induced in populations of
AL cells in which 5, 10, 20, or 100% of the cells had received
exactly one particle through the nucleus is shown in the upper
curve of Fig. 1. The mutant fractions (MF) predicted, assuming
no bystander interaction between the irradiated and nonirradiated
cells, are shown in the lower curve. The experimental curve
is significantly different from that expected. For example, the
mutant fraction when 5% of the cells had been irradiated was
58% of that when all of the cells were irradiated (induced mutant
fractions were 57 and 98 per 105 survivors, respectively). It is of
interest to note that there was little change in the yield of mutants
when the fraction of irradiated cells increased from 10 to 100%.
This result could be a reflection that the percentage of irradiated
cells in the population that were in direct contact with non-hit
cells in mediating the bystander response had reached a plateau
at 10% level and that further increases in the proportion of
irradiated cells would not enhance the bystander response.
Because the range of secondary electrons from particles of this
energy is 0.25 m (28), it is highly unlikely that direct radiation
damage to the nontargeted cells by secondary electrons contributes
to the bystander effect.
Involvement of Gap Junction-Mediated Cell–Cell Communication in
Bystander Mutagenesis. Because a high cell density implies cell–
cell contact in the process, we investigated the relationship
between gap junctional activity and particle-induced bystander
mutagenic effect in two ways: (i) the use of octanol to inhibit gap
junction-mediated intercellular communication (21) and (ii) the
use of genetically engineered cells that lack gap junctions. In our
first set of studies, we treated AL cells with a nontoxic and largely
nonmutagenic dose of octanol (1 mM) beginning 2 h before and
until 3 days after irradiation. As shown in Fig. 2, octanol reduced
the yield of induced CD59 mutants from 92 35 to 16 3 per
105 survivors. Treatment of octanol alone resulted in an induced
mutant fraction of 10 4 per 105 survivors. Although this
result indicates a role of gap junctions in the bystander mutagenic
response, the effects of octanol are not limited to gap
junctions but can affect other cellular structures and functions,
including membrane fluidity (29). Therefore, to investigate more
specifically the role of gap junction-mediated cell–cell communication
with particle-induced bystander mutagenicity, we
used cells in which gap junctional activity was suppressed by a
dominant negative connexin construct.
AL-AH1-9 Cells Genetically Deficient in Connexin 43 Demonstrate No
Bystander Mutagenesis. We transfected AH1-9 cells with either a
connexin 43 overexpressing or a dominant negative construct. By
using the standard scrape-loading test as a measure of gap
junction activity (22), we found that the migration of Lucifer
yellow was completely blocked in cells carrying the dominant
negative connexin 43 vector (Fig. 3B, DN6). In contrast, the dye
was found to migrate many cell layers in distance among cells
carrying connexin 43 overexpressing construct (Fig. 3A, CX10).
Parental AL cells and AH1-9 cells showed a moderate migration
of Lucifer yellow (data not shown). Significantly, AH1-9 cells
containing the connexin 43 expressing vector showed a higher
bystander mutagenic yield than that of vector control (Fig. 4,
Table 1). In contrast, there was little, if any, bystander effect
among AH1-9 cells carrying the dominant negative vector (Table
1). These data clearly show that gap junction intercellular
communication is critical in mediating the bystander mutagenesis,
although the nature of the signaling molecules involved in
the communication between particle-traversed and nontraversed
cells remains to be established.
Using Chromatid Breakage as an Endpoint To Assess the Bystander
Effect. In addition to gene mutations, chromosomal aberrations
are an important class of DNA damages induced by particles.
Therefore, we further compared the incidence of chromatid-type
breaks induced in AL cells where a single particle was delivered
to the nucleus in either 20 or 100% of the cultures. As shown in
Fig. 5B, in population where every cell had been irradiated, 93%
of the cells contained three or more chromatid breaks. This
result was in sharp contrast to the control where only 10% of the
cells contained one break (Fig. 5A). When 20% of the cells in a
population were irradiated with a single particle, 75% of the cells
were expected to contain no breaks if one assumed there was no
interaction between the irradiated and nonirradiated cells (Fig.
5C). In actuality, only 36% of the cells in this population showed
no chromatid breaks (Fig. 5D). Furthermore, the profile of
chromatid breaks was very different from that in which 100% of
the cells in the population were hit. Addition of octanol (1 mM)
Fig. 1. Induced CD59 mutant fractions per 105 survivors obtained from
populations of AL cells in which 0, 5, 10, 20, or 100% had been irradiated with
exactly one particle through its nucleus. Induced mutant fraction total
mutant fraction minus background incidence, which was 4610 mutants per
105 clonogenic survivors in AL cells used in these experiments. Data are pooled
from three to seven independent experiments. Error bars representSD. The
calculated curve deviates slightly from a straight line fitting because of the
slight cytotoxic effect of single particle traversal among the irradiated cells.
Fig. 2. Effect of octanol treatment (1 mM, 2 h before and maintained until
3 days after irradiation) on mutant fractions of AL cell population of which
20%had been irradiated with a single particle through the nucleus. Data are
from three independent experiments. Error bars represent SD.
14412 www.pnas.orgcgidoi10.1073pnas.251524798 Zhou et al.
or lindane (40 M) to the 20% irradiated culture likewise
obliterated the increase in chromatid breaks resulting from the
bystander effects to a profile similar to that shown in Fig. 5C
(data not shown).
Discussion
Both epidemiological and experimental animal studies have
indicated an association between exposure to radon ( particles)
and the incidence of lung cancer (see ref. 30 for review).
Although the mechanisms of radiation carcinogenesis have not
been elucidated, there is good evidence that radiation-induced
genetic changes such as chromosomal aberrations, gene mutations,
and genomic instability play a critical role in the
process. Because direct epidemiological studies on indoor
radon exposure and lung cancer are equivocal, risk for low
level of exposure received by the general population have been
based on extrapolation from higher exposures in studies of
underground miners assuming a linear, no-threshold doseresponse
relationship. This model for cancer estimation, however,
has been a subject of controversy for decades because
there is insufficient observational basis to confirm the model
(3, 31). Moreover, application of this model as a basis of
radioprotection and risk assessment by no means signifies its
validity, merely a precautionary necessity.
It has always been accepted that most of the deleterious
effects of ionizing radiation including particles are attributable
to direct nuclear hits. Recent evidence, however, indicates
that extranuclear or extracellular events are also important
in mediating the genotoxic effects of particles. Early
investigations of the radiation-induced bystander effect measured
the frequency of sister chromatid exchanges (SCE) in
populations of cells exposed to low fluencies of particles. It
was found that SCE levels were significantly higher than
expected from target theory calculations of the number of cells
that had actually been hit by an particle (4, 6). Furthermore,
such biological effects as induction of micronuclei (10), gene
Fig. 3. The scrape-loading assay (20) was used to evaluate levels of gap
junction-mediated intercellular communication inAL-AH1-9-transfected cells.
In connexin 43 overexpressing (CX10) cells (A), Lucifer yellow migrated a
distance of several cell layers away from the scrape. In contrast, in cells
transfected with the dominant negative vector (DN6) cells (B), there was no
migration of the dye (200).
Fig. 4. Mutation fraction (MF) from population of AL-AH1-9 cells transfected
with connexin 43 overexpressing vector (CX10), a dominant negative connexin
43 vector (DN6), or vector alone (CXV2). Data are from three to four independent
experiments. Error bars represent SD. The populations of AH1-9 cells
used in these experiments have higher mutant induction as well as background
mutant level than the parental AL cells.
Zhou et al. PNAS December 4, 2001 vol. 98 no. 25 14413
CELL BIOLOGY
mutation (12, 13), and expression of stress-related genes (5, 9,
15) can occur in a significantly higher proportion of cells than
in those traversed by an particle. There is also evidence that
bystander effects are involved in malignant transformation of
mammalian cells in vitro (16).
By using a precision charged-particle microbeam, we reported
that cells that had been lethally irradiated with particles could
induce mutagenesis in neighboring cells not directly hit by the
particles, and that mutant induction depended on cell–cell
communication (13). However, exposure to high dose of
particles is an unlikely scenario in environmental exposures to
radon. To extend this observation, we show here that a single
particle traversal of a small fraction ofAL cells (10–20%) induces
a mutagenic response similar to that occurring when 100% of the
cells in the population are hit, and that gap junction-mediated
cell–cell communication plays an important role in the process.
Although it is not clear whether directly irradiated cells are
equally responsive to the bystander effect observed in nonirradiated
cells, it is likely that, when cells are directly hit, they
initiate a series of self-preservation mechanisms including DNA
repair and a cell-signaling process that diminish their ability to
respond to bystander signaling. In other words, irradiated cells
behave differently from bystander, nonirradiated cells in their
collective response to mutagenic signals.
Two important questions need to be addressed: (i) What are
the mechanism(s) of the bystander mutagenic process, and (ii)
what is the implication of the present findings to low dose
radiation risk assessment? Based on the literature, it is likely that
at least two pathways are involved in mediating radiationinduced
bystander effects (see ref. 32 for review). In sparsely
populated cultures, any induction of a bystander response clearly
requires the presence of oxyradicals or other soluble mediators
(10). In contrast, studies (including ours) with confluent monolayers
have implicated gap junctional activities (12–15). These
latter findings are consistent with our result that certain cytotoxic
factor(s), such as cytokines and reactive oxygen or nitrogen
species released into the culture medium from irradiated cells,
have little, if any, effect on bystander mutagenesis (33). Furthermore,
pretreatment of cells with the intracellular radical
scavenger, N-acetyl cysteine (10 mM) had little effect on bystander
mutagenic yield (data not shown). However, there is
evidence that, among confluent human fibroblast cultures, secretion
of cytokines or other growth-promoting factors by irradiated
cells leads to enhanced production of reactive oxygen
species in bystander cells (6, 7). These two observations among
confluent cultures are not necessarily mutually exclusive because
there is evidence that radiation induces long-lived organic
radicals that persist for hours (34).
We also found that the gap junction inhibitor octanol significantly
decreased the mutant yield. These results were further
confirmed in transfected cells carrying a dominant negative
connexin that reduced the mutation frequency of the cell
Fig. 5. Induction of chromatid-type breaks per cell from populations of AL cells in which 0, 20 or 100% of cells were traversed by exactly one particle through
the nucleus. The data are from three to four independent experiments. Bars represent SD.
Table 1. Comparison of mutant fractions (MF) in population of
AL-AH1-9 cells transfected with either connexin 43
overexpressing vector (CX10), dominant negative
vector (DN6), or with vector alone (CXV2)
Cell line
Level of
GJCC* Induced MF
† Predicted MF
MF because
of bystander‡
CX10 High 467 97 370
CXV2 Moderate 346 123 223
DN6 None 149 141 8
*GJCC, gap junction-mediated cell–cell communication.
†Mutant fraction, number of CD59 mutants per 105 survivors.
‡Bystander mutagenic yield induced MF minus predicted MF, assuming no
interaction between irradiated and nonirradiated cells.
14414 www.pnas.orgcgidoi10.1073pnas.251524798 Zhou et al.
populations to the level expected assuming no bystander effect.
The gap junction channels have an apparent selectivity based
principally on molecular size, allowing the movement of molecules
smaller than 1,000 Da, such as cAMP, but preventing the
movement of proteins or nucleic acids. These findings show that
gap junction intercellular communication plays a critical role in
bystander mutagenesis when cells are in close contact, although
the nature of the signaling molecules involved in the communication
between particle-traversed and nontraversed cells remains
to be established.
Our G2 PCC studies indicated that bystander effect could also
be demonstrated at the chromosomal level. Compared with the
traditional mitotic preparation based on metaphase spread, the
use of chemically induced PCC provides a more sensitive and
easier approach to score chromatid damage in mammalian cells
(26). For example, in Calyculin A-treated cells, theG2 PCC index
was found to be seven times higher than the mitotic index after
a comparable treatment with colcemid (26). It should be noted
that the profile of chromatid breaks found in populations where
100% of cells were hit was very different from that in which only
20% of cells received a hit. The findings are consistent with the
observations that (i) high linear-energy-transfer radiation produces
multiple damaged sites in the nucleus of hit cells (35) and
(ii) the type of mutations induced as a result of the bystander
effect is qualitatively different from that of direct nuclear hit (12,
13). The findings that, in the presence of octanol or lindane, the
profile of chromatid breaks in this latter population resembled
that found in controls suggest that gap junctions are indeed
involved in the bystander phenomenon.
Our studies provide clear evidence that a single particle can
induce mutations and chromosome aberrations in cells that
received no direct radiation exposure to their DNA. These
findings imply that the target for radiation-induced genetic
damage is larger than an individual cell. The observation is
important in formulating risk assessment models because, for
particles, a cell cannot receive a dose lower than a single traversal
and these hit cells are a minority population in lung tissue
exposed to environmental radon. The observation that irradiation
of as few as 10% of a cell population results in a mutagenic
yield similar to that when all of the cells in the population are hit
indicates that low dose particle irradiation can induce a huge
bystander mutagenic response in neighboring cells not directly
traversed by particles. The genotoxic risk at such a low dose
region, therefore, may be significantly underestimated based on
current practice. Because radiation-induced bystander response
(mainly cell killing and genomic instability) has been demonstrated
with low linear-energy-transfer radiation such as X- or
-rays (8, 32), our findings may not be inconsistent with the
recent study of radiation-related cancer risk among A-bomb
survivors at the dose range of 0.15–0.30 Sv (36). Results of our
present studies cast doubt on the dose at which dose linearity
would be expected, a strong indication that the models presently
used in predicting radiation risk at low doses are inadequate and
need to be reexamined.
We thank Drs. Eric Hall and Charles Geard for helpful discussion and
Dr. Hiroshi Yamasaki for providing the dominant negative connexin 43
plasmids. This work was supported by National Cancer Institute Grants
CA75384, 49062, and 36447 and by National Cancer Institute Research
Resource Grant RR 11623.
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