Mustonen et al. BMC Cell Biology (2018) 19:17
https://doi.org/10.1186/s12860-018-0169-9

RESEARCH ARTICLE

Open AccessFibroblasts from bank voles inhabiting
Chernobyl have increased resistanceagainst oxidative and DNA stresses
Venla Mustonen1, Jenni Kesäniemi1, Anton Lavrinienko1, Eugene Tukalenko2, Tapio Mappes3, Phillip C. Watts1
and Jaana Jurvansuu1*

Abstract
Background: Elevated levels of environmental ionizing radiation can be a selective pressure for wildlife by
producing reactive oxygen species and DNA damage. However, the underlying molecular mechanisms that are
affected are not known.
Results: We isolated skin fibroblasts from bank voles (Myodes glareolus) inhabiting the Chernobyl nuclear power
plant accident site where background radiation levels are about 100 times greater than in uncontaminated areas.
After a 10 Gy dose of gamma radiation fibroblasts from Chernobyl animals recovered faster than fibroblasts isolated
from bank voles living in uncontaminated control area. The Chernobyl fibroblasts were able to sustain significantly
higher doses of an oxidant and they had, on average, a higher total antioxidant capacity than the control
fibroblasts. Furthermore, the Chernobyl fibroblasts were also significantly more resistant than the control fibroblasts
to continuous exposure to three DNA damaging drugs. After drug treatment transcription of p53-target gene proapoptotic Bax was higher in the control than in the Chernobyl fibroblasts.
Conclusion: Fibroblasts isolated from bank voles inhabiting Chernobyl nuclear power plant accident site show
elevated antioxidant levels, lower sensitivity to apoptosis, and increased resistance against oxidative and DNA
stresses. These cellular qualities may help bank voles inhabiting Chernobyl to cope with environmental radioactivity.
Keywords: Bank vole, Chernobyl, Environmental ionizing radiation, p53, DNA damage, Antioxidant capacity

Background
The Chernobyl nuclear power plant disaster in 1986 wasclassified by the International Atomic Energy Agency to themost severe radiation accident level. Since then, the mostcontaminated area around the nuclear power plant has
been closed to the general public. This Chernobyl exclusionzone covers about 2600 km2 and still contains patches ofradioactively contaminated soil emitting from normal background levels of 0.2 μSv/h up to about 200 μSv/h [1].
A meta-analysis has shown that ionizing radiation
has increased mutation frequency in various Chernobyl taxa as represented by, for example, discolorations,cataracts, chromosomal abnormalities, or cancers [2].
* Correspondence: jaana.jurvansuu@oulu.fi
1
Department of Ecology and Genetics, University of Oulu, FI-90014 Oulu,
Finland
Full list of author information is available at the end of the article

Another meta-analysis on wildlife inhabiting Chernobyl
area found a small to intermediate increase in oxidative
damage (such as imbalance between oxidants and antioxidants) and a decrease in antioxidant defenses (such as depleted antioxidant levels) [3]. However, the prevalence of
these effects varied among species, implying that there is
no standard response to chronic low-dose radiation andthat some species are more radioresistant than others. Accordingly, the species abundance in Chernobyl area has
been reported to be in decline, constant, and on rise, due toeither the adverse effects of ionizing radiation or because oflack of detrimental human activity [4–7]. Furthermore, evidence for an adaptive response to chronic low-dose radiation at Chernobyl is somewhat equivocal [8]. Nonetheless,adaptation has been reported in plants: plants within Chernobyl area have higher resistance to DNA-damaging chemicals, antioxidants, osmotic stress, and gamma irradiation© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Mustonen et al. BMC Cell Biology (2018) 19:17

than control plants and have also changes in antioxidant
and stress responses, and in DNA repair [9–11].
The bank vole (Myodes glareolus) is an ideal mammalianmodel to study the effects of environmental radiation in
Chernobyl, because it is common at the site, remains close
to the surface source of radiation, and as a small animal
has relatively small territory. Bank vole population sizes
were very low following the accident at Chernobyl but
numbers of this species rebounded during the following
year, probably via immigration from several nearby populations [12, 13]. Since the accident, Chernobyl bank voles
have been shown to harbor slight but significant increasein amounts of chromosomal aberrations, mitochondrial
DNA mutations, and cataracts in females, yet other work
has found no signs of genotoxic stress [14–17].
Ionizing radiation can cause long-lasting and inherited effects by damaging DNA directly or via water-derived free
radicals. To determine whether exposure to chronic
low-dose radiation extending possibly several generations has affected bank vole cells’ ability to tolerateoxidative and DNA stress we isolated skin fibroblasts
from bank voles from the Chernobyl exclusion zone
and from an uncontaminated control area near Kiev.
We chose to study skin fibroblasts because they are easy
to isolate even in field conditions. The fibroblasts werechallenged with gamma radiation to study their reactions
to the original type of stress, and with an oxidative agent
and three DNA damaging drugs to dissect the responses
to two biologically important aspects of ionizing radiation:oxidative stress and DNA damage. In all treatments, Chernobyl fibroblasts were, on average, able to sustain the insults better than the control fibroblasts. Moreover,Chernobyl fibroblasts had higher total antioxidant capacity than the control cells and were less sensitive to DNA
damage induced lethality, both of which processes may
explain their increased resistance against radiation.

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scarcely. Recovery date was scored when cells reached100% confluency. The delay of recovery (Fig. 1) was calculated by how many days longer the irradiated cell line took
to reach confluency in comparison to the untreated cellline. Fibroblasts from Chernobyl animals were significantly(Student’s t-test p-value = 0.028) faster to recover after theirradiation than fibroblasts from control animals. Control
fibroblasts took on average 9.2 days longer to fill the plateafter 10 Gy irradiation whereas Chernobyl cells took just
6.8 days longer than the corresponding untreated cell lines.
Chernobyl fibroblasts grow in higher concentrations ofoxidant and have higher total antioxidant capacity thancontrol fibroblasts

To determine whether Chernobyl fibroblasts are more resistant to the oxidative or the DNA damaging effect of radiation we first studied the effect of the oxidant tert-butylhydroperoxide on the cells. Cells were plated on 24 wellplates at 80% confluency and exposed to different concentrations (50–150 μM) of tert-butyl hydroperoxide for 24 h
before changing the media. Cells were allowed to grow fora week and then 100% confluent wells were scored(Fig. 2a). Chernobyl fibroblasts were able to recover andgrow in about 40 μM higher concentration of the oxidant
than the control fibroblasts (xCTRL = 66.7 μM, xCHERNOBYL = 108.3 μM, Student’s t-test p-value = 0.0096). We also
tested whether the cells would differ in response to constant exposure to small concentrations (10–50 μM) of theoxidant added every other day for four times but bothResults
Chernobyl fibroblasts recover after 10 Gy irradiation
faster than control fibroblasts

Skin fibroblasts were isolated from eight male bank volesfrom the Chernobyl exclusion zone (an average dose rateof 21 μSv/h) and from an uncontaminated control area
near Kiev (an average dose rate of 0.2 μSv/h). The same
number of fibroblasts for each cell line were irradiated at
the rate of 3.5 Gy/min from a cesium-137 source. The
cells were allowed to recover for 24 h and then replated toa flask with about 4.5 times bigger growth surface area.
After 24 h of the irradiation, we did not see increase in
apoptotic or floating dead cells in any of the cell lines andthus we chose a growth based assay instead of a viability
test to study the cells. A clonogenic survival assay, typicalfor these kind of experiments, was not suitable for theseprimary fibroblasts because they readily die if plated too

Fig. 1 Chernobyl bank vole fibroblasts recover faster after irradiation
than control cell lines. The fibroblast cell lines isolated from
Chernobyl bank voles (pink) were able to reach 100% confluency, on
average, 2 days faster than the control cell lines (grey) after 10 Gy
irradiation. “The delay of recovery” represents how many days longer
an irradiated cell line took to cover the plate than thecorresponding non-irradiated cell line. The figure shows results fromthree separate experiments using eight Chernobyl (N = 24) and eightcontrol (N = 24) cell lines. In the box blot the box presents the lower
and upper quartiles, median is indicated by a line, whiskers show
values within 1.5 interquartile range from the boxed values, andcircles are outliers (R version 3.3.3)

Mustonen et al. BMC Cell Biology (2018) 19:17

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Fig. 2 Chernobyl fibroblasts survive in higher oxidant concentration and have more antioxidants than control fibroblasts. a Fibroblasts isolated
from Chernobyl bank voles were, on average, able to grow in about 40 μM higher concentration of oxidizing agent tert-butyl hydroperoxide(TBH2O2) than control bank vole fibroblasts. Cells were treated with different concentrations of the oxidant for 24 h after which fresh cell culture
media was changed and cells were let grow for 7 days before scoring the wells that remained 100% confluent. b. Chernobyl fibroblasts havesignificantly higher total antioxidant capacity in untreated and oxidant-treated cells than control fibroblasts. For oxidant treatment cells weregrown in 25 μM of TBH2O2 for 24 h before the total antioxidant capacity was measured. Results are from three separate experiments using eight
Chernobyl (N = 24) and eight control (N = 24) cell lines, variation is shown by standard deviation, and statistical analysis was done with Student’s
t-test (* = p ≤ 0.05, ** = p ≤ 0.01)

groups, except one control cell line, were able to adjust
and grew in all the concentrations (Additional file 1).
To assess if Chernobyl fibroblasts tolerate more oxidantbecause they had more antioxidants, total antioxidant levels
were measured by cell extracts’ capacity to reduce copper.
Cell extracts from cells grown in normal conditions or after
24 h challenge with 25 μM of tert-butyl hydroperoxide we
tested (Fig. 2b). On average, Chernobyl fibroblasts had almost twice the total antioxidant capacity in normal cell culture conditions than the control cells (xCTRL = 3.5 nM/μg,
xCHERNOBYL = 6.5 nM/μg, p-value = 0.010). Total antioxidant capacity in the oxidant-challenged cell lines was
greater than in unchallenged cell lines in both groups andthe increase was similar: 2.4 and 2.2 units for control andChernobyl cells, respectively. The similar level of antioxidant induction in the two groups suggests that regulatory
responses in Chernobyl fibroblasts were intact. However,Chernobyl fibroblasts had still significantly higher total antioxidant capacity (p-value = 0.041) than the control cells
after the antioxidant treatment. The higher basal antioxidant levels in Chernobyl fibroblasts may protect the cells
against excess oxidants generated by ionizing radiation
exposure.
Chernobyl fibroblasts grow in higher concentrations of
DNA damaging drugs than control fibroblasts

We tested the bank vole fibroblasts’ ability to withstandDNA damage by exposing cells to DNA cross-linking
drug cisplatin, DNA-intercalating drug doxorubicin, andtopoisomerase inhibitor etoposide. Cisplatin adducts arerepaired mainly by nucleotide excision repair whereas
doxorubicin and etoposide produce double-strand breaks
that are subject to homologous recombination [18]. Cells
were plated at 80% confluence and treated with different
concentrations of the drugs for 24 h before replacing themedia. The cells were allowed to grow for a week before
scoring the concentration where they were able to grow to
100% confluency. Chernobyl fibroblasts tolerated significantly more of etoposide than the control cells (xCTRL =44.1 μM, xCHERNOBYL = 83.6 μM, Student’s t-test p-value =0.004; Fig. 3c). Yet there was no significant differences
between the groups to the other drug treatments(Fig. 3a and b). Because a constant small amount ofDNA damage is more appropriate condition to mimic theenvironmental radiation to which the Chernobyl bankvoles are exposed, we treated the cells with low concentrations of the drugs every other day for four times and recorded the 100% confluent wells a day after the lasttreatment. Chernobyl cells were significantly more resistant to all DNA damaging drugs (Fig. 3d to f), especiallydoxorubicin and etoposide (Fig. 3e and f), than the control
cells. On average, the Chernobyl fibroblasts were able togrow in over four times higher doxorubicin concentrations
than the control cell lines (xCTRL = 2.2 μM, xCHERNOBYL =9.0 μM, p-value < 0.0001). The difference in tolerance between Chernobyl and control cells treated with etoposide(xCTRL = 14.6 μM, xCHERNOBYL = 27.5. μM, p-value = 0.0015;
Fig. 3f) and cisplatin (xCTRL = 13.1 μM, xCHERNOBYL =Mustonen et al. BMC Cell Biology (2018) 19:17

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D

E

F

G

H

I

Fig. 3 Chernobyl bank vole fibroblasts survive on higher concentrations of DNA damaging drugs than control cells. Cells were plated at
80% confluency and treated with different concentrations of the DNA damaging drugs for 24 h after which fresh cell culture media waschanged and the cells were let to grow for 7 days before scoring the wells that were 100% confluent. Treatment with a.) cisplatin or b.)
doxorubicin did not show significant difference between the Chernobyl and control cell lines, whereas Chernobyl cells were able to growin, on average, about 30 μM higher concentrations of c.) etoposide. For constant exposure of DNA damaging drugs cells were treatedwith the drug every other day for four times before scoring the wells that were 100% confluent. Chernobyl fibroblasts were able to growin significantly higher chronic concentrations of D.) cisplatin, e.) doxorubicin and f.) etoposide than control cells. Examples from one
experiment (N = 8) on the effect of constant drug treatment on reduction of 100% confluent Chernobyl and control fibroblast cultures
after treatment with g.) cisplatin, h.) doxorubicin, and i.) etoposide. Results, except g-i, are from three separate experiments using eight
Chernobyl (N = 24) and eight control (N = 24) cell lines, variation is shown by standard deviation, and statistical analysis was done with
Student’s t-test (ns = p > 0.05, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001)

17.2 μM, p-value = 0.018; Fig. 3e) were smaller but still significant. Chernobyl cells seemed more tolerant to dsDNA
break producing drugs doxorubicin and etoposide than tocisplatin-derived adducts. In contrast to oxidant exposure,cells were incapable of adjusting to constant dose of DNA
damaging drugs: the longer the cells were treated with thedrugs, the more sensitive they became as shown by thechronic dose experiment where the 100% confluent wells
were recorded a day after every treatment (Fig. 3g to i).
After DNA damage Chernobyl fibroblasts remain arrested
at G2 whereas control fibroblasts die

Chernobyl fibroblasts might withstand higher concentrations of DNA damaging drugs because they either hadenhanced DNA repair capacity or were able to arrest
longer to repair the damage. We tested the fibroblasts’ability to repair DNA damage by host-cell reactivation
assay on oxidized, nicked, and linearized plasmid substrates. Our results showed that there was no significant
difference in repair efficiencies between Chernobyl andcontrol fibroblasts (Additional file 2). Next, we tested thecell cycle effects of etoposide-mediated DNA damage on
the fibroblasts. We treated cells with 20 μM of etoposidefor 24 h and collected samples 24, 48, and 72 h from thestart of the treatment for propidium iodide flow cytometry. Most of the cells arrested at the G2 phase (with 2 N
DNA content) within 24 h after treatment. The control
cells started to die (sub-G1) visibly more 2 days after theMustonen et al. BMC Cell Biology (2018) 19:17

treatment (Fig. 4a and b). Etoposide is known to cause
apoptosis [18] and according to Annexin V staining thiswas also observed in bank vole fibroblasts (Additional file 3).
On average, Chernobyl fibroblasts were able to stay arrested
at G2 longer than the control cell lines and thus avoiding
cell death (Fig. 4c). Significantly more control cells weredying already 24 h after the treatment (xCTRL = 6.5%, xCHERNOBYL = 3.0%, Student’s t-test p-value = 0.001) and after 2days there was, on average, about three times as many control cells dying as Chernobyl cells (xCTRL = 17.5%, xCHERNOBYL = 6.9%, p-value = 0.00008). There were no significant
differences between the cell lines in amount of cells
remaining at G1 (i.e., with 1 N DNA content) at any time
point. The control cells’ inability to remain at G2 phase together with the observation of their increased cell deathsuggests that Chernobyl fibroblasts may have an intact
DNA damage signaling pathway that leads to cell cycle arrest and lower sensitivity to apoptosis.
After DNA damage p53-target transcripts are induced
more in control than in Chernobyl fibroblasts

Activation of transcription factor p53 by DNA damage canlead to apoptosis, senescence or cell cycle arrest. We collected etoposide-treated (20 μM of etoposide for 24 h) samples 48 and 72 h post-treatment for cDNA preparation. We
quantified the transcript levels of p53-target genes: cell
cycle regulators p21 and Gadd45α, pro-apoptotic proteins
Puma and Bax, and p53-inhibitor Mdm2 and standardized
them to beta-actin transcripts. Results are shown as foldchange from untreated sample (Fig. 5). Induction of Baxand Mdm2 was significantly higher at both time
points in control fibroblasts in comparison to Chernobyl cells (p-valueBAX-48h = 0.0021, p-valueBAX-72h =0.00036, p-valueMDM2-48h = 0.0099, p-valueMDM2-72h =0.00096; Fig. 5c and e). P21 was significantly more induced in control than Chernobyl cells at 48 h (p-value =0.028; Fig. 5a) and Gadd45α at 72 h time point (p-value =0.0016, Fig. 5b). No significant differences in Puma induction levels were detected at either time point (Fig. 5d).
The amount of pro-apoptotic Bax transcript induction
was about 20% higher in control than in Chernobyl celllines, which is consistent with the higher percentage ofdying control cells seen in the flow cytometry experiment.

Discussion
Fibroblasts from Chernobyl bank voles were, on average,
more resistant to radiation, oxidant treatment and DNA
damaging drugs than the control fibroblasts. The DNA
damaging drugs we used, etoposide, doxorubicin, and cisplatin have all been suggested to cause oxidant byproducts[19–21], thus the higher antioxidant levels of the Chernobyl fibroblasts’ may have also protected them against theindirect effects of these DNA damaging drugs. Neither
control nor Chernobyl cell lines were homogenous in theirPage 5 of 10

responses to any of the experimental treatments; there
were always one or two moderately resistant control andsensitive Chernobyl cell line, as is evident from the large
variance of the experiments. This variability may indicatethat the resistant traits are naturally occurring genetic variations. Bank vole populations at Chernobyl are from manydifferent source populations and thus do host much genetic
variation that may provide useful adaptive traits [12, 22].
Our results suggest that the environmental radiation in
Chernobyl may select for adaptive cellular qualities inbank voles that defend against oxidants and help recoverfrom DNA damage. However, statistical support for population level adaptation would require larger sample sizes
from several different bank vole populations. Nevertheless,no-one has yet presented changed nucleotide substitution
rates in the nuclear genomes of any Chernobyl inhabitingorganisms. Mutations, as manifested by chromosomal abnormalities and phenotypic changes, have been reported
in diverse Chernobyl taxa [2]. Although Baker et al. [17]have shown increased mutation rate of mitochondrial genomes in bank voles inhabiting Chernobyl, the results are
not directly applicable to the nuclear genome because ofdifferences in the DNA repair mechanisms and oxidative
processes and conditions between nucleus and mitochondria. We found that Chernobyl fibroblasts withstood especially well chronic doses of dsDNA break inducing drugs(doxorubicin and etoposide) and, unlike control fibroblasts, they were able to stably arrest after DNA damageat G2 phase, when homologous recombination, the mostaccurate mechanism to repair dsDNA breaks, is most active. Thus, the implication of our results is the potential
for bank vole cells to mount an efficient cellular mechanism against DNA breaks produced by radiation.
Chernobyl fibroblasts had more antioxidants in standard cell culture conditions than control cells and they
were able to grow in higher oxidant concentrations.
Chernobyl bank voles’ darker fur was hypothesized byBoratyński et al. [23] to be indicative of preferential production of eumelanin instead of pheomelanin, of whichbiosynthesis reduces reserves of antioxidant glutathione.
Galván et al. [24] showed that Chernobyl birds living inareas of high radiation have increased glutathione levels,
which correlated positively with improved body condition and reduced DNA damage. The antioxidant levels
in Chernobyl birds have also been reported to be depleted, possibly due to the chronic radiation exposure
leading to increased amounts of oxygen radicals [25, 26].
The unchallenged antioxidant levels were not tested inthese ecological studies, but the results do suggest thatantioxidants are one of the key factors in response
against environmental radiation. Cells raise antioxidant
levels following oxidant exposure and this reaction has beenshown to be a crucial cellular response against low-dose radiation [27, 28]. Consequently, priming cells with oxidant

Mustonen et al. BMC Cell Biology (2018) 19:17

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Fig. 4 After DNA damage Chernobyl fibroblasts arrest stably at G2 phase. Example propidium iodide flow cytometry profile for one a.) control
and b. Chernobyl cell line after 24, 48, and 72 h from etaposide treatment. c. The bar charts show the percentage of cells in G1, G2, and sub-G1
cell cycle phase for Chernobyl and control fibroblasts according to their DNA content by flow cytometry after 24, 48, and 72 h from etoposidetreatment. The location of cells in sub-G1 (dying cells), G1, and G2 cell cycle phase are marked in the DNA histogram. The bar chart results arefrom three separate experiments using eight Chernobyl (N = 24) and eight control (N = 24) cell lines, variation is shown by standard deviation,and statistical analysis was done with Student’s t-test (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001)

Mustonen et al. BMC Cell Biology (2018) 19:17

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Fig. 5 P53-target genes are induced more strongly in control cells
than in Chernobyl bank vole fibroblasts. RNA was collected from
untreated and etoposide-treated (20 μM) cells 48 and 72 h after the
drug treatment. CDNA was prepared and quantitative-PCR was run
with gene specific primers, and transcript levels were standardized
to beta-actin transcript. Results are shown as a fold increase from
untreated samples for a.) p21, b.) Gadd45α, c.) Bax, d.) Puma, and e.)
Mdm2. The results are from three separate experiments using theeight Chernobyl (N = 24) and eight control cell lines (N = 24),
variation is shown by standard deviation, and statistical analysis wasdone with Student’s t-test (** = p ≤ 0.01, *** = p ≤ 0.001)

B

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D

E

or irradiation that increases antioxidant levels has shown toprotect cells from a second exposure to radiation [27, 29].
Our results show that Chernobyl bank vole fibroblasts seemto be primed against abnormally high levels of oxidants.
P53 is a transcription factor and its activity (or lack ofit) after genotoxic stress often determines whether thecell will arrest, commit apoptosis, or proceed into senescence (or mitotic catastrophe). P53 is activated by phosphorylation and the length and level of the activation
together with other regulatory factors and cellular condition will decide cell fate. Low-dose chronic radiation has
been shown to activate p53 and induce premature senescence in primary endothelial cells and fibroblasts [28, 30].
After p53 activating etoposide treatment Chernobyl cells
were able to arrest stably at G2 whereas control cells
started to die. Our results suggest that the higher induction levels of pro-apoptotic Bax were sufficient to induce
apoptosis in control cell lines yet the lower levels of cell
cycle regulators, p21 and Gadd45α, were high enough for
the Chernobyl fibroblasts to remain at G2. Similar changes
in amounts of p53-target gene transcripts have had a major
impact on cell death in cancer cell lines with mutated p53[31, 32]. Furthermore, p53 has been shown to have a rolealso in population level adaptation of mole rats to hypoxic
environments [33]. However, in 1999 DeWoody [34] examined a short segment of Chernobyl bank voles’ p53 geneand found no apparent genetic changes in the sequence.

Conclusions
Compared to control fibroblasts, fibroblasts isolated frombank voles inhabiting Chernobyl nuclear power plant accident site have elevated basal antioxidant levels and show
decrease susceptibility to apoptosis after DNA damaging
treatment. Accordingly, the Chernobyl fibroblasts weremore resistant against oxidative and chronic DNA stresses.
Environmental radiation research is complicated by numerous interactions between exposed animal and its exposed environment. Meta-analysis has shown thatorganisms inhabiting Chernobyl area are approximately
eight times more sensitive to radiation than species exposed
to similar levels of radiation in controlled laboratory experiments [35]. Thus, the harmful effects of radiation apparently escalate with increased biological interaction andMustonen et al. BMC Cell Biology (2018) 19:17

complexity. These ecological interactions might explain
why some bank voles inhabiting Chernobyl nuclear accident site have two cellular mechanisms, high antioxidant
levels and insensitivity to apoptosis, to cope with seemingly
minor excess of environmental radiation.

Methods
Cells

Cells were isolated from eight male bank voles from the
Chernobyl exclusion zone (GPS coordinates N51.44536
E30.06522) and eight male voles from Kiev area
(N50.525859 E30.436933) (Additional file 4). Bank voles
were caught using Ugglan Special2 multiple-capture live
traps (Grahnab, Sweden) during June of 2016. The levelof soil radiation at the trapping sites were measured at
one cm above the ground using a Geiger-Mueller
dosimeter (Inspector, International Medcom INC). Selected bank voles were of similar size (length: xCTRL =24.5 cm, xCHERNOBYL = 21.9 cm, Student’s t-test p-value= 0.06; weight: xCTRL = 88.7 g, xCHERNOBYL = 92.1 g Student’s t-test p-value = 0.08). Animals were euthanized bycervical dislocation and all the procedures were performed in accordance with international guidelines andregulations for the use of animals in research. The study
was approved by the Finnish Ethical Committee (license
number ESAVI/7256/04.10.07/2014).
Skin fibroblasts were isolated 1–4 h after the animals
were sacrificed according to the protocol of Seluanov [36].
In short, about one cm2 area of shaved skin from armpitof the animal was clipped and put into PBS for 10 min
and then cut into small pieces and incubated at 37 °C in
DMEM (30%) / PBS (70%) / Liberase DL Research Grade
Enzyme (500 μg/ml) (Sigma-Aldrich) for 30 min. The tissue was filtered through 70 μm nylon mesh and placed
into cell culture tube with DMEM (high glucose, GlutaMAX™) / 20% FBS / penicillin (100 U/ml) / streptomycin(100 μg/ml) / amphotericin B (1 μg/ml) (all from Thermofisher) and incubated at 37 °C for 2 days.
Cells were routinely cultured at 36 °C / 80% humidity /5% CO2 in DMEM / 10% FBS / penicillin / streptomycin /amphotericin B. All experiment were done with similar
passage numbers for both control and Chernobyl cells between passages 5–30. After cell passage 30 some of thecell lines showed chromosomal number abnormalities.
Cell treatments

For cell treatments, 20,000 cells were plated to 24 wellplates and let grow for 1 day to about 80% confluency before the treatment. Tert-butyl hydroperoxide (Alfa Aesar)
was diluted in PBS and added to cells at concentrations of
150, 125, 100, 75, and 50 μM for one dose experiments
and at 50, 40, 30, 20, and 10 μM for long exposure. Cisplatin (Sigma-Aldrich) was solubilized into 0.90% NaCland added to cells at concentrations of 50, 40, 30, 20, andPage 8 of 10

10 μM for one dose experiments and at 25, 20, 15, 10, and5 μM for long exposure. Doxorubicin (Sigma-Aldrich) was
solubilized into DMSO and added to the cells at concentration of 160, 80, 40, 20, 10 μM for one dose experiments
and at 20, 15, 10, 5, 1 μM for long exposure. Etoposide(Cayman Chemical) was solubilized into DMSO andadded to cells at concentrations of 200, 100, 50, 25, and12.5 μM for one dose experiments and at 45, 35, 25, 15,
and 5 μM for long exposure. For one dose experiments
the cells were incubated in the drug for 24 h after which
fresh media was changed and cells were let to recover for
7 days before scoring the 100% confluent wells. For longer
exposures, cells were treated with the drug every otherday for four times by removing the old media and adding
a new drug dose in fresh media. The 100% confluent
wells were scored a day after the last treatment. Experimental controls were treated only with the drug
solubilization solution.
For irradiation 20,000 cells were plated to cell culture
tubes (NuncTM) and irradiated the next day with 10 Gy at
the rate of 3.5 Gy/min from Cesium-137 source (Gammacell 2000). The cells were allowed to recover from the irradiation for 24 h and then replated to 25 cm2 plates. Cells
were allowed to grow until they were 100% confluent.
Total antioxidant assay

For each cell line, 40,000 cells were plated on 6 well plates
and allowed to grow for 2 days before adding tert-butylhydroperoxide for final concentration of 25 μM (or thesame volume of PBS to experimental controls). Total antioxidant levels were measured 24 h after the treatment
with OxiSelectTM Total Antioxidant Capacity Assay kit(Cell Biolabs) and the protein concentration was measured with Bio-Rad Protein Assay.
Host-cell reactivation assay

Five thousand cells were plated on 96 well plate, treatednext day with 20 μM etoposide for 8 hours, and then transfected with pGL3 (Promega) plasmid treated either with
Nb.BsmI that nicked the plasmid coding sequence threetimes, with HindIII that linearized the plasmid after promoter sequence, or with 50 μM FeSO4 and 1 mM H2O2,
which created oxidative damage on the plasmid. To control
transfection efficiency cells were transfected also with
pNL1.1 nano-luc vector. Luciferase expression was analysed
24 h after transfection with Nano-Glo Dual-Luciferase reporter assay system as suggested by the manufacturer(Promega).
Quantitative-PCR

Total RNA was isolated with TRIzol® Reagent (Thermofisher) as suggested by the manufacturer. CDNA was prepared from 0.5 μg of RNA using RevertAid H Minus First
Strand cDNA Synthesis Kit (Thermofisher) with randomMustonen et al. BMC Cell Biology (2018) 19:17

primers as suggested by the manufacturer. Quantitative-PCR was done with 5xHOT FIREPOL® Evagreen®
qPCR Mix Plus (SolisBiodyne) as suggested by the
manufacturer. Primers were designed to span at least
one intron and were as follows; beta-actin: forward
5’ TGCGTGACATCAAAGAGAAG and reverse 5’GA
TGCCAGAAGATTCCATA, p21: forward 5’CTCCTG
TGGGCACTTTAGGG and reverse 5’TGTCGCTGT
CCTGCACTCT, Gadd45α: forward 5’CTGGAGGAAGTGCTCAGCAA and reverse 5’GGTCGACATTGAGCAGCTTG, Bax: forward 5’TGCCCGAGTTAATCAGAACCA and reverse 5’GGACTCCAGCCACAAAGATAGT, Puma: forward 5’CGGAGACAAGAAGAGCAGCA and reverse 5’TAGTTGGGCTCCATTTCGG
G, Mdm2: forward 5’CAGTCTGAGTGAAGATGGG
CA and reverse 5’AGCTAAGGAGATCTCAGGGT
CT, p53: forward 5’CCAACACAAGCTCCTCTCCC
and reverse 5’ATTCGCGTCCTGAGCATCC (metabion international AG and Eurofins Genomics). For
each gene the PCR product was cloned into
pJet-plasmid as recommended by CloneJET PCR
Cloning kit manual and used in qPCR to make astandard curve. Results were analyzed with LightCycler® 96 system software.
Propidium iodide flow cytometry

Forty thousand cells were plated on 6 well plates andtreated with 20 μM etoposide the next day. 24 h after thetreatment fresh cell culture media was changed and firstsamples were collected to 70% EtOH. Similarly 48 h and
72 h post-treatment samples were collected and stored inethanol. Before the flow cytometry cells were resuspended
into propidium iodide (100 μg/ml) and RNAse A (100 μg/ml) (Thermofisher). For each sample 10,000 cells were recorded with FACSCalibur. Flow cytometry results wereanalyzed with Flowing Software 2.5.1.

Additional files
Additional file 1: Chernobyl and control fibroblasts are able to adjust to
constant exposure to small concentrations of oxidant. The oxidant wasadded every other day for four times before scoring the wells that were100% confluent a day after the last exposure. The results are from three
separate experiments using the eight Chernobyl (N = 24) and eightcontrol cell lines (N = 24). Variation is shown by standard deviation.
(PDF 28 kb)
Additional file 2: The repair efficiency of nicked, oxidized, or linear
plasmids is similar in control and Chernobyl bank vole fibroblasts. For
host-cell reactivation assay, 5000 cells were plated on 96 well plate,treated next day with 20 μM etoposide for 8 hours, and then transfectedwith pGL3 (Promega) plasmid treated either with Nb.BsmI that nicked theplasmid coding sequence three times, with HindIII that linearized theplasmid after promoter sequence, or with 50 μM FeSO4 and 1 mM H2O2,
which created oxidative damage on the plasmid. To control transfection
efficiency cells were transfected also with pNL1.1 nano-luc vector. Luciferase expression was analysed 24 h after transfection with Nano-Glo DualLuciferase reporter assay system as suggested by the manufacturer

Page 9 of 10(Promega). The bar charts show the relation of standardized treated tostandardized untreated plasmid expression. The results are from four separate experiments using the eight Chernobyl (N = 32) and eight control
cell lines (N = 32). Variation is shown by standard deviation. (PDF 27 kb)
Additional file 3: Etoposide induces apoptosis in bank vole fibroblasts.
We treated the cells with DMSO or 20 μM of etoposide for 24 h, replaced
the media, and collected samples 72 h post-treatment for propidium iodide and Annexin V flow cytometry with eBioscience Annexin V apoptosis
Detection kit FITC as recommended by the manufacturer. The figure
shows one control and one Chernobyl cell line. The percentage ofhealthy cells are shown in the lower-left corner, necrotic cells in theupper-left corner, and apoptotic cells at right. (PDF 66 kb)
Additional file 4: The trapping locations of the bank voles used in thisstudy for fibroblast isolation. The green circles present the location at
Kiev control area (average site radiation 0.2 μSv/h) where the control
voles were trapped and the red circle denotes the site where Chernobyl
voles were caught (average site radiation 21 μSv/h). Black dashed line
indicates the 30 km Chernobyl exclusion zone. CNPP with a red triangle
shows the location of the Chernobyl nuclear power plants. A map of
Ukraine as an inset show by a red square the location of Chernobyl area.
Map was created with ESRI ArcGIS 10.0. Satellite imagery © CNES/Airbus
DS, Earthstar Geographics. Source: Esri, DigitalGlobe, GeoEye, i-cubed,
Earthstar Geographics, CNES/Airbus DS, USDA, USGS, AEX, Getmapping,
Aerogrid, IGN, IGP, swisstopo, and the GIS User Community | Esri, HERE,
DeLorme. (PDF 106 kb)
Acknowledgements
We are grateful to Virpi Glumoff (University of Oulu) for allocating facilities
for the cell culture work and guidance with the flow cytometry and AnnaLeena Manninen, Miika Nieminen, Ilpo Pelkonen, and Eeva-Riitta Savolainen(University of Oulu, University Hospital of Oulu, and Nordlab) for access tothe cell irradiator. We are also grateful to Iryna Kozeretska (Taras Shevchenko
National University of Kyiv) for the laboratory facilities at Ukraine. We acknowledge Gennadi Milinevsky, Igor Chizhevsky, Kati Kivisaari, and Zbyszek
Boratyński for help in organizing fieldwork in Ukraine. We thank reviewers fortheir constructive comments.
Funding
This research was supported by a Kone Foundation grant for JJ. The funding
body had no role in in the design of the study and collection, analysis, andinterpretation of data or in writing the manuscript.
Availability of data and materials
The datasets used and analysed during the current study are available fromthe corresponding author on reasonable request. Materials are available on
reasonable request.
Authors’ contributions
VM Performed qPCR and Annexin V FACS analysis and contributed to thewriting of the manuscript. JK designed qPCR experiments and contributed to
the writing of the manuscript. AL designed and performed on site animalcollection and contributed to the writing of the manuscript. ET performed
the on site animal collection and animal treatments. TM established thestudy site and methods in Ukraine and contributed to the writing of themanuscript. PCW Designed the on site animal collection and contributed to
the writing of the manuscript. JJ Designed and performed the experiments,
interpret and analyzed the results, and wrote the manuscript. All authors
read and approved the final manuscript.
Ethics approval
All procedures complied with the legal requirements and internationalguidelines for the use of animals in research. All necessary permissions forthe experiments were obtained from the Animal Experimentation
Committee (permission no. ESAVI/7256/04.10.07/2014).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.

Mustonen et al. BMC Cell Biology (2018) 19:17

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.
Author details
1
Department of Ecology and Genetics, University of Oulu, FI-90014 Oulu,
Finland. 2Institute of Biology and Medicine, Taras Shevchenko National
University of Kyiv, Kyiv UA-03022, Ukraine. 3Department of Biological andEnvironmental Science, University of Jyväskylä, FI-40014 Jyväskylä, Finland.
Received: 11 May 2018 Accepted: 20 August 2018

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