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DOI 10.1393/ncb/i2010-10889-y
Basic topics: Applied Physics
IL NUOVO CIMENTO Online First
Effects of deprivation of background environmental radiation on
cultured human cells
M. C. Carbone(1)(∗),M.Pinto(1)(4)(7)(*)(∗∗), F. Antonelli(1)(4)(7),
M. Balata(3),M.Belli(4)(7), L. Conti Devirgiliis(2),O.Sapora(5),
G. Simone(4)(7),E.Sorrentino(4)(7),M.A.Tabocchini(4)(7)andL. Satta(6)
(1)Museo Storico della Fisica e Centro Studi e Ricerche “Enrico Fermi”
piazza del Viminale 2, 00184, Rome, Italy
(2)Dipartimento di Biologia di Base e Applicata, Universit`a dell’Aquila - Via G. Falcone 25
67100 Coppito (AQ), Italy
(3)INFN, Laboratori Nazionali del Gran Sasso - S.S. 17 BIS km. 18.910, 67010 Assergi (AQ)
Italy
(4)Dipartimento di Tecnologie e Salute, ISS - Viale Regina Elena 299, 00161 Rome, Italy
(5)Dipartimento di Ambiente e Prevenzione Primaria, ISS - Viale Regina Elena 299
00161 Rome, Italy
(6)INFN, Laboratori Nazionali di Frascati - via E. Fermi 40, 00044 Frascati (RM), Italy
(7)INFN, Gr.coll. Sanit`a, Sezione di Roma1 - Viale Regina Elena 299, 00161 Rome, Italy
(ricevuto il 12 Gennaio 2010; approvato il 21 Aprile 2010; pubblicato online il 12 Luglio 2010)
Summary. — In this paper we present results from an experiment aimed at investi-
gating whether living cells are influenced by background ionizing radiation. Parallel
human cell cultures were set-up in two separate laboratories and maintained for sev-
eral months under identical conditions but for a 80×different level of background
ionizing radiation. Periodically, the cell cultures were monitored for the onset of di-
vergences in biochemical behavior, using two distinct cellular biology assays, namely
micronuclei induction and activity of enzymes implicated in the management of ox-
idative stress. To reveal any subtle modifications, responses were also amplified
by subjecting cell cultures to acute stress induced by exposure to moderately high
doses of ionizing radiation. Compared to reference radiation background condi-
tions, cultures maintained in a reduced background radiation environment handled
the consequences of acute stress with diminished efficacy.
PAC S 87.53.-j – Effects of ionizing radiation on biological systems.
PAC S 87.50.-a – Effects of electromagnetic and acoustic fields on biological sys-
tems.
(∗) MCC and MP contributed equally in this work.
(∗∗) E-mail: pinto@centrofermi.it
c
Societ`a Italiana di Fisica 1
2M.C. CARBONE, M. PINTO, F. ANTONELLI, ETC.
1. – Introduction
Life has evolved on Earth for 3 billion years in the presence of background ionizing
radiation. Living organisms may keep memory of being in the presence of ultra low dose
rate radiation, so that a question arises as to whether the biochemical behavior of living
organisms and life on Earth would differ in the absence of radiation [1,2].
To address this scientific question, an ideal experimental design would consist in the
twin set-up of a cell or animal culture in a laboratory where background radiation is
reduced as low as possible, and in a reference laboratory at “normal” background levels.
Using a number of experimental assays, the experimenter would monitor the biologi-
cal model for the onset of any differential behavior between the two laboratories. To
achieve a condition of reduced environmental radiation, a suitable location is the under-
ground laboratory of the Italian National Institute for Nuclear Physics at Gran Sasso
(LNGS/INFN), located in central Italy alongside a highway tunnel between the cities of
L’Aquila and Teramo, underneath at least 1400 m of limestone rock. This unique setting
allows a significant reduction in the muon flux [3] and the virtual elimination of atmo-
spheric showers [4]. Inside the underground laboratory, the organic nature of limestone
rock results in a greatly reduced environmental background radiation [5]; moreover, the
laboratory walls are lined with specifically chosen low-activity concrete. To minimize
accumulation of 222Rn which would result in a substantial exposure to densely ioniz-
ing, and therefore highly biologically effective, radiation, the underground laboratory is
equipped with a ventilation system that captures air from outside the highway tunnel
and pumps it inside the laboratories. Occasional and short-term controlled shut-down of
such ventilation system can result in spikes of 222Rn activity.
In this work, the “reference” laboratory was established at Istituto Superiore di Sanit`a
near central Rome, such that the reduction of environmental radiation level achieved,
relative to the reference laboratory, is 70-fold, but reaches 80-fold when a low-activity
iron shielding is adopted inside the underground laboratory [6].
Unlike most physics experiments, our biophysics experiment is focused on measure-
ments on biological, living systems. The terrific complexity of a living being, even at the
cellular level, affects the accuracy of any experimental determination. To minimize the
effects of the large number of variables involved, one needs to define a suitable experi-
mental model which offers control over the variable of major interest, while attempting
to diminish the role of potential confounders. For preliminary investigations in which one
needs to isolate and focus on few variables, in vitro cell culture systems are typically at
an advantage over more complex animal models, and we have accordingly designed and
conducted a series of experiments in several in vitro models [7, 8]. Our previous experi-
ments suggested that cells grown in reduced background radiation conditions manifested
an altered response to acute doses of ionizing radiation or exposures to genotoxic agents
such as methyl-methan-sulphonate (MMS) [7, 8]. Specifically, cells grown in reduced
background radiation conditions were less capable of facing the damage induced by such
agents, relative to cultures maintained in reference background conditions (outdoors).
Markers of such deviations included gene mutations, DNA damage as measured by mi-
cronuclei induction [9], activity of enzymes involved in the management of oxidative
stress [10-12], and cell cycle duration.
Stemming from our past experience, we have recently set up an in vitro experiment
whereby parallel cultures of human lymphoblastoid TK6 cells were maintained in iden-
tical experimental conditions with the exception of background radiation. As our ear-
lier measurements revealed different responses between underground and above-ground
EFFECT OF ENVIRONMENTAL RADIATION ON HUMAN CELLS 3
cultures of S. Cerevisiae and Chinese hamster V79 cells only after several cell genera-
tions [7,8], we set out to carry on the human lymphoblastoid cultures for up to continuous
12 months in both laboratories [6].
At regular time intervals, the on-going cell cultures were subjected to multiple mea-
surements, to detect any signs of biochemical deviations due to different levels of envi-
ronmental radiation, in terms of duplication time, ability to cope with endogenous DNA
damage via a “micronucleus” assay, and ability to maintain redox homeostasis via an
enzymatic activity assay. Additionally, at regular time intervals, cells were frozen in
liquid nitrogen (LN2) for archival of biological specimens.
Since subtle biological effects were expected after such a minimal variation of living
environment, which could remain undetected by our measuring apparatus, we also set to
implement a potentially more sensitive detecting test by challenging the twin cell cultures
with an acute exposure to a cytotoxic agent. In so doing, it is expected that any small
biological differences could be amplified and detected. Conveniently, this cytotoxic agent
was ionizing radiation itself, imparted at doses about three orders of magnitude larger
than exposures caused by environmental radiation.
While the experiment is still in progress, we here report on preliminary measure-
ments performed after six months of continuous cell culture in both laboratories, hinting
at significant effects of deprivation of background radiation. We observed lower repair
efficiency of DNA damage caused by acute exposure of radiation in the underground, en-
vironmental radiation-deprived cultures, and altered management of redox environment
as a function of background radiation levels.
2. – Material and methods
Experimental methods were described in detail elsewhere [6]. Briefly, human lym-
phoblastoid TK6 cultures were maintained in standard conditions at 37◦C in a cell
culture CO2incubator for up to six months. All plastic and reagents were kept identical
at both cell culture sites to minimize any laboratory effect on the responses sought. To
control for any genetic drift that could result, during the six months of continuous cul-
ture, in the isolation of genetic mutants that may bias the experimental observations, we
set up two independent cell cultures at each laboratory site, and conducted all experi-
ments on each “sister” cell culture separately and concomitantly. With this approach, it
is highly unlikely that genetic drift could have acted in the same fashion in both sister
cultures.
For challenge experiments, exposures to acute doses of ionizing radiation were carried
out at an external facility in the city of L’Aquila. In these instances, exposure to normal
environmental background radiation, including cosmic radiation, was negligible.
All cultures were handled similarly and experimental assays were run on coded sam-
ples, with the code unknown to the person that conducted the terminal measurements.
3. – Assessment of chromosomal damage
Some DNA damage events can lead to morphological changes in the nuclear DNA that
may be readily visualized using conventional fluorescence microscopy techniques. When a
damaged DNA fragment, detached from a chromosome, is produced, it may be lost from
the nucleus when a cell attempts to undergo mitotic division. The micronucleus assay [9]
allows the visualization of these events by blocking cell division before such fragments
go lost. At the microscope, the detached DNA fragment will appear as a fluorescent
4M.C. CARBONE, M. PINTO, F. ANTONELLI, ETC.
Fig. 1. – A typical image captured at the fluorescence microscope, showing two nuclei of a
duplicating cell with a “micronucleus”.
spot, similar to a cell nucleus, but about 1/10 of its size, positioned nearby two regular
nuclei which the cell was attempting to segregate into two daughter cells (fig. 1). The
frequency of micronucleated events, i.e. of binucleated cells that contain at least one
micronucleus, will serve as a quantitative indicator of failed repair of damage to DNA
caused by a genotoxic agent, in our case ionizing radiation. The experimental protocol
for preparation and scoring of micronuclei events was described elsewhere [6,9].
4. – Measurements of activity of enzymes involved in the management of
reactive oxygen species
Regular cell functions require maintenance of several balance states. One of such
equilibrium states is that of chemical species that are involved in reduction and oxidation
of other species. Deviations from a state of equilibrium may generate a range of cellular
effects. In particular, accumulation of reactive oxygen species (ROS) can be readily
generated by acute exposures to ionizing radiation and the efficacy of their clearance
is an indicator of a cell’s health. An established method to evaluate the efficacy of
ROS clearance is the direct measurement of the activity of enzymes implicated in ROS
management. In particular, we have measured the enzymatic activity of superoxide
dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) using methods
that have been described elsewhere [6]. Since the aforementioned enzymes act in concert,
measurements of enzymatic activity are also accompanied by activity ratios that provide
additional indication of ROS scavenging efficacy.
5. – Results
5.1. Chromosomal damage. – The frequency of micronuclei was determined for all
the cultures maintained for six months under reduced and reference background radia-
tion (two independent determinations, hereon denominated “runs”), as well as for the
cell culture that was used to generate the previous two when the experiment was first
EFFECT OF ENVIRONMENTAL RADIATION ON HUMAN CELLS 5
Fig. 2. – Frequency of micronuclei events in basal (A) and 2 Gy-irradiated cell cultures (B) main-
tained for six months under reduced (LNGS) and reference (ISS) background radiation, as well
as for the cell culture that was used to generate the previous two when the experiment was first
set up (t0). Error bars represent standard errors, assuming Poisson’s statistics. Experimental
runs have been indicated with numbers 1 and 2 in the legend of panel B.
set-up (denominated “t0”). Contingency tables were built, and subjected to a χ2test,
to verify whether measurements from independent samples were not statistically differ-
ent, in which case they were cumulated to increase precision. This was the case for the
basal frequency of micronuclei events (an estimate of lack of repair or mis-repair of spon-
taneous chromosomal damage) for both cultures maintained in reduced and standard
background environment. Measurements indicate an increase with time in both labo-
ratories compared to the initial level (t0value; panel A, fig. 2). However, this increase
is statistically significant only for the cell culture maintained for 6 months in reference
radiation background conditions (ISS, p=0.009). These data show that cells main-
tained under reduced background conditions for 6 months display a limited increase of
spontaneous level of DNA damage, relative to cells maintained in reference conditions.
Maintenance of cell cultures in reduced background environment may cause subtle
effects that could remain undetected by this technique. To amplify any such effects, we
used the same technique to determine the micronuclei frequency in cells that were exposed
to an acute dose of 2 Gy of ionizing radiation, i.e. large enough to produce extensive DNA
damage [13]. The results are reported in fig. 2 (panel B). For acutely irradiated cultures,
micronuclei frequency determinations were not cumulated from independent runs (p>
0.05). Results indicate that maintenance of cells under reduced background radiation
environment increases the frequency of micronuclei that is caused by the acute radiation
exposure, relative to our reference background radiation conditions (first run 1.45 ±0.09
6M.C. CARBONE, M. PINTO, F. ANTONELLI, ETC.
Fig. 3. – Ratios of Se-GPx to SOD measured activity, expressed in arbitrary units, for basal
and 1 Gy-irradiated cell cultures maintained for six months under reduced (LNGS) and reference
(ISS) background radiation, as well as for the cell culture that was used to generate the previous
two when the experiment was first set-up (t0). Error bars represent standard errors.
fold, p<0.05; second run 1.29±0.09 fold, p<0.05). Moreover, the micronuclei frequency
measured in cells maintained under reference background conditions is consistent with
that measured at t0(first run 1.03 ±0.09 fold, p>0.05; second run 0.90 ±0.08 fold,
p>0.05). Given that the amount of DNA damage caused must be identical (the damage
caused by the low level exposure to background ionizing radiation being negligible), this
enhancement in micronuclei frequency reflects a reduced efficacy of DNA repair activity.
Such effect may have been caused by the reduction of background radiation. Taken
together and albeit the variability across the two experimental runs, this experimental
evidence shows that, at the level of repair of DNA damage, the behavior of the cells
maintained under reduced radiation background for prolonged times is different from
that of reference cultures.
5.2. Management of reactive oxygen species. – Measurements of SOD, CAT, and GPx
activities were carried out on both cell cultures, maintained for six months under the
two different background radiation environments and on the t0culture, before and after
irradiation with a dose of 1 Gy. The GPx/SOD and CAT/SOD ratios may be used
as estimators of the protection level against imbalance of ROS. A large value of any of
these two ratios will indicate that the cell cultures are able to react against the potentially
detrimental effects of excess ROS. By contrast, low values of these ratios indicate reduced
ability to face ROS imbalances.
In fig. 3 we present only the measured GPx/SOD ratios for all cultures used in this
experiment. Relative to the t0cultures, cells maintained for prolonged times in culture
show an altered capacity to control cellular ROS equilibrium. Specifically, the response
of t0cultures to acute exposure to ionizing radiation results in a reduced ROS scavenging
activity, relative to constitutive/basal levels, while maintenance of cultures for six months
in reference background conditions (ISS) results in the opposite effect. By contrast, such
response to ionizing radiation was significantly lost in cultures chronically deprived of
background radiation (LNGS).
6. – Discussion
As part of an on-going experiment, we have here reported the results of several
measurements conducted on human TK6 cell cultures maintained for six months under
EFFECT OF ENVIRONMENTAL RADIATION ON HUMAN CELLS 7
reduced or reference background radiation environments. We hypothesized that attenua-
tion of background radiation levels may cause subtle biochemical changes in cell cultures,
and we have therefore elected to conduct a highly controlled experiment in which all ma-
terials and methods were rigorously identical with the exception of background radiation
levels. Given that six months of continuous culture results in hundreds of mitotic cell
divisions, genetic drifts may have occurred in these cultures that could have rendered
them differently able to respond to the experimental stimuli that we chose to employ.
To control for any such drift, we set up two independent cell cultures at each laboratory
site, and conducted all experiments on each “sister” cell culture separately and concomi-
tantly. With this approach it is highly unlikely that genetic drift could have acted in the
same fashion to both sister cultures. In fact, our measurements indicated that responses
of sister cultures were the same, within statistical errors (not shown), so that we here
presented cumulated results within each run (figs. 2 and 3).
Compared to reference background conditions (ISS), cultures maintained in a reduced
background radiation environment (LNGS) handled DNA damage caused by acute expo-
sure to ionizing radiation less efficiently (fig. 2) and were unable to react to the imbalance
of ROS that follows acute exposure to 1 Gy (fig. 3).
At the level of DNA damage, basal levels of micronuclei frequencies were significantly
larger in ISS cultures compared to t0cells (two standard deviations), while basal levels
were unchanged, within errors, in LNGS cultures (fig. 2, panel A). Such difference may
be caused by either an increase in the spontaneous amount of DNA damage in ISS
cultures, via an augmented oxidative stress, or by an increased efficiency of the DNA
repair machinery in the LNGS cultures. This second hypothesis is less plausible, since it
hinges on the idea that a reduced level of damage could stimulate repair.
Acute exposures to ionizing radiation showed larger levels of micronuclei frequency in
LNGS cultures than in ISS cultures, whose micronuclei frequency are the same, within
errors, as those measured in t0cultures (fig. 2, panel B). As indicated above, constitutive
oxidative stress caused by background ionizing radiation could be responsible for these
observations: at such extremely low levels, oxidative stress originating from background
radiation may be exerting a stimulating action on the DNA repair machinery, which,
upon virtual elimination of the stimulus, as in the LNGS cultures, may be not prepared
to face the consequence of exposure to a much larger dose of radiation.
At the level of ROS management in response to acute exposure to ionizing radi-
ation (fig. 3), our measurements indicated that the cellular scavenging efficiency (as
measured via the Se-GPx/SOD ratio) is diminished in t0cells, while it is elevated in
cultures maintained for six months in reference radiation background conditions. These
measurements are the result of five independent repeats and show differences larger
than two standard deviations; it is unlikely that they were due to chance. However,
the emerging results are puzzling and difficult to interpret. We hypothesize that the
cultures maintained for six months in reference radiation background conditions have
elevated ROS levels, compared to t0cultures. Although a different behavior is not
manifest at the level of constitutive ROS scavenging activity (fig. 3), a constitutively
high level of ROS may render ISS cultures more prone to react to the sudden eleva-
tion of ROS levels that results from the acute exposure to ionizing radiation. Such
hypothesis of elevated constitutive ROS levels in the ISS cultures is attractive, as it
could also explain the results of the measurements made with the micronucleus assay
(fig. 2, panel A) as discussed earlier. Interestingly, other investigators have demon-
strated that alterations of ROS levels may have significant impact on basic cellular
functions [14].
8M.C. CARBONE, M. PINTO, F. ANTONELLI, ETC.
By contrast, the metabolic behavior of cells maintained for six months in reduced
background radiation environment is different both from the t0cultures and from the
ISS cultures, as warranted by relative small experimental errors on these measurements.
As a matter of fact, the response of LNGS cultures to acute radiation at the level of ROS
management, within experimental errors, was negligible. These differences prompt us to
conclude that the ISS and LNGS cultures have acquired a markedly diverging behavior
(more than three standard deviations).
Taken together, experimental measurements at the level of DNA damage and repair,
and management of ROS balance, strongly suggest that TK6 cell cultures develop differ-
ent behaviors under reduced or reference background radiation environments. Therefore,
it is likely that the presence of a normal background radiation environment favors the
maintenance of protective responses in human TK6 cells. It is tempting to speculate
that evolution of life on Earth may have been different in the absence of environmental,
background ionizing radiation [1].
High throughput comparative analyses of the cellular transcriptome (i.e. the set of
all RNA molecules) of cells chronically maintained under reduced background radiation,
and reference conditions, are in progress using DNA microarrays and will be subject of
a future report.
∗∗∗
FA, MCC, and MP are grateful to Prof. A. Zichichi and to the “Museo Storico della
Fisica e Centro Studi e Ricerche Enrico Fermi” (Cosmic Silence project) for awarding
their junior investigator fellowships. All the authors are grateful for the support given
by the Cosmic Silence project.
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