Comparative Biochemistry and Physiology, Part C 151 (2010) 271–277

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Comparative Biochemistry and Physiology, Part C
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p c

DNA damage in barn swallows (Hirundo rustica) from the Chernobyl region detectedby use of the comet assay
Andrea Bonisoli-Alquati a,⁎, Andrew Voris b,e, Timothy A. Mousseau c, Anders Pape Møller d,f,
Nicola Saino a, Michael D. Wyatt ba

Dipartimento di Biologia, Sez. Ecologia, Università degli Studi di Milano, via Celoria 26, I-20133 Milano, Italy
Department of Pharmaceutical and Biomedical Sciences, University of South Carolina, Columbia, SC 29208, USA
Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USAdLaboratoire d'Ecologie, Systématique et Evolution, CNRS UMR 8079, Université Paris-Sud, Bâtiment 362, F-91405 Orsay Cedex, FranceeMedical University of South Carolina, Charleston, SC 29425, USAf
Center for Advanced Study, Drammensveien 78, NO-0271 Oslo, Norwaybca r t i c l e

i n f oArticle history:
Received 4 August 2009
Received in revised form 18 November 2009
Accepted 19 November 2009
Available online 24 November 2009
Keywords:
Barn swallow
Chernobyl
Comet assay
Genetic damage
Hirundo rusticaa b s t r a c t
We investigated levels of DNA damage in blood cells of barn swallows (Hirundo rustica) inhabiting theChernobyl region to evaluate whether chronic exposure to low-level radioactive contamination continues to
induce genetic damage in free-living populations of animals. Blood samples were obtained from barn swallows
collected at sites with different background levels of radiation, including a relatively uncontaminated area. The
extent of DNA damage was evaluated using the alkaline (pH = 12.1) comet assay, a robust and sensitiveelectrophoresis-based technique widely employed in research ranging from biomonitoring to clinical studies.
We found that levels of DNA damage, as indexed by the extent of DNA migration, were increased in barnswallows living in areas surrounding Chernobyl when compared to swallows sampled at low-level sites. The
results we obtained are consistent with previous ﬁndings on this same species, which showed that swallows
breeding in areas heavily contaminated with radionuclides have increased mutation rates, higher oxidative
stress and incidence of morphological aberrations and tumors. Overall, these results indicate that chronic
exposure to radioactive contaminants, even 20 years after the accident at the Chernobyl nuclear power plant,
continues to induce DNA damage in cells of free-living animals.
© 2009 Elsevier Inc. All rights reserved.

1. Introduction
The accident at the Chernobyl nuclear power station in 1986 likely
represents the worst accidental release of radionuclides (mainly
131
Iodine, 90Strontium and 137Caesium; Shestopalov, 1996) into theenvironment. Although the subsequent exposure to radiation isconsidered to have been at a low level (i.e. b20 mSv/year; Golikov
and Balonov, 1993), harmful biological consequences spanning fromthe individual to the community and ecosystem level caused by theradioactive contamination have been demonstrated in a number ofstudies (review in Møller and Mousseau, 2006).
The barn swallow (Hirundo rustica), a small, long-distant migratory
passerine bird, has been a model species for many of these studies,owing to its thoroughly investigated ecology and life history. Studies
conducted on barn swallows have shown that individuals breeding incontaminated areas around Chernobyl experience reduced survival rate
and reproductive success, as demonstrated by both a lower proportion⁎ Corresponding author. Tel.: +39 0250314716.
E-mail address: andrea.bonisoli@unimi.it (A. Bonisoli-Alquati).
1532-0456/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpc.2009.11.006

of reproducing adults and an increase in hatching failure compared tobirds breeding in uncontaminated areas (Møller et al., 2005a). In birds
from the Chernobyl area, individual lymphocyte counts and immunoglobulin concentrations are reduced (Camplani et al., 1999) andantioxidants, such as carotenoids and vitamins A and E, in plasma andliver are seriously decreased (Møller et al., 2005b). Partial albinism inthe plumage, together with other morphological abnormalities, is also
more frequent in birds breeding in the most heavily contaminated areasand is associated with a reduction in individual survival rates (Ellegren
et al., 1997; Møller et al., 2007). Consistent with this decrease in ﬁtness,barn swallow population sizes in the Chernobyl region are declining(Ellegren et al., 1997) despite immigration from populations inuncontaminated areas (Møller et al., 2006).
At the genetic level, barn swallows from around Chernobyl have agermline mutation rate enhanced by a factor of two to ten, compared
to a Ukrainian and an Italian control population, as revealed by meansof a parent–offspring regression analysis at both expressed and noncoding (i.e. microsatellite) loci (Ellegren et al., 1997). Moreover, in astudy where abnormal sperm were used to track individual mutations, the increase in the germline mutation rate was related toincreased levels of background radiation (Møller et al., 2005b). In

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addition, this study provided support to the hypothesis of an inverse
relationship in this species between the level of antioxidants in an
individual bird and sensitivity to radiation (i.e. increases in mutation
rate among individuals may be due to decreased levels of circulatingor stored antioxidants) (Møller et al., 2005b).
Ionizing radiation can induce DNA damage either by direct effects(Iliakis, 1991) or indirectly by producing an excess of free radicals that
can in turn damage DNA (Imlay and Linn, 1988; Imlay et al., 1988).
Antioxidant compounds provide protection against the free radicals (i.e.
reactive oxygen species, ROS) that arise as an unavoidable byproduct of
mitochondrial respiration; ROS can cause signiﬁcant damage tobiological molecules, including DNA (Halliwell and Gutteridge, 2007).
Induction of ROS by radiation would reduce the available antioxidant
levels, which might increase the risk for DNA damage resulting fromfurther exposure to ROS (Neyfakh et al., 1998a,b; Kumerova et al., 2000;
see Møller et al., 2005b).
Several techniques have then been developed that allow for thequantiﬁcation of the extent of DNA damage induced by genotoxic agentsin vivo (Tice et al., 2000). Here, we report the ﬁrst ﬁnding of an increase
in DNA damage in a wild population of any animal after the Chernobyl
disaster by using the alkaline (pH = 12.1) single-cell gel electrophoresis
assay (or comet assay), a technique designed to detect the ability ofchemical and physical agents to induce increased levels of DNA strand
breaks in eukaryote cells (Tice et al., 2000). The comet assay is usuallyregarded as being one of the most sensitive and reliable techniques, withadvantages of cost-effectiveness, speed, and the fact that observations
are made at the level of the individual cell (Tice et al., 2000). Due to theseadvantages and to its applicability to virtually any eukaryotic cell, theuse of the comet assay has recently been extended to a wide range ofapplications, spanning from the molecular to the epidemiological level(Collins et al., 1997; see reviews in Fairbairn et al., 1995; Collins, 2004;
Tice et al., 2000; Jha, 2008).
This technique has traditionally been used to investigate radiationinduced DNA damage in humans and other model species (reviews in
Tice and Strauss, 1995; Olive, 1999). Recently, this technique isincreasingly being applied to ecogenotoxicological studies of wildorganisms (Kleinjans and van Schooten, 2002), in both animal andplant species (Jha, 2004, 2008; Dhawan et al., 2009; Frenzilli et al.,2009), usually for investigating the effects of chemical pollutants andpredict the genotoxic potential of chemicals. Studies on birds are
relatively rare compared to other taxonomic groups (Pastor et al.,2001a,b, 2004).
The comet assay has also been used to assess levels of DNA damage
in people, mainly children and clean-up workers, inhabiting contaminated areas near Chernobyl (Frenzilli et al., 1998, 2001; Garcia andMandina, 2005). To the best of our knowledge, however, the cometassay has not previously been used to examine DNA damage in cells of
any wild population exposed to the radioactive contaminants in theenvironment following the accident at Chernobyl in 1986.
2. Material and methods
2.1. General ﬁeld procedures
During June 2005–2007, we captured barn swallows using mist
nets across open doors and windows in farm buildings previously
inspected for their presence. This method has been shown to beunbiased and highly efﬁcient during the breeding season (Møller andSzép, 2002). The individuals included in this study were captured in
Dytiaku (51° 07′ N, 30° 09′ E), Pysky (51° 05′ N, 30° 03′ E), Vesniane(51° 18′ N, 29° 37′ E), and Ghovtnere (50° 12′ N, 30° 49′ E) (Fig. 1).
Blood samples were taken by puncturing the brachial vein, collecting
the blood in heparinized capillary tubes.
Background radiation levels were measured using a hand-held
dosimeter (Inspector, SE International, Summertown, TN, USA). Basedon our ﬁeld measurements, we classiﬁed sampling sites as low-,intermediate or high-level. Background levels of radiation were0.02 μSv/h in the low-level site (Ghovtnere), 0.05 μSv/h in both theintermediate-level sites (Dytiaku and Pysky) and 2.9 μSv/h in thehigh-level site (Vesniane) (see Fig.1). Field-collected radiation
measurements were highly correlated with ofﬁcially published data
(Shestopalov, 1996; see Møller et al., 2008a for details).
2.2. The comet assay
Blood samples were processed according to the protocol by Singhet al. (1988) with minor modiﬁcations. Samples were ﬁrst frozen at−20 °C and later transferred to a −80 °C freezer. The same procedure
was applied to samples from all years. Samples were quickly thawed byimmersion in a 37 °C water bath, to avoid consolidation of microcrystals of ice into larger forms, that are known to be damaging (Mazuret al., 1981). Erythrocytes were suspended in Ca2+/Mg2+-free PBS at
2 × 105 cells/mL. A volume of this suspension was mixed with 1% lowmelting agarose (LMA) in a 1:10 ratio, and layered on two-well,precoated slides (Trevigen, Gaithersburg, MD, USA). Slides were placed
at 4 °C in the dark for 30 min, then immersed in ice-cold, freshly
prepared lysing solution (Lysis Solution, Trevigen, pH= 10.0, combinedwith 10% dimethyl sulfoxide (DMSO)), and allowed to sit in the dark for
1 h. DNA unwinding was carried out in a tray ﬁlled with freshly preparedalkaline solution (1 mM ethylenediaminetetraacetic acid disodium salt
(Na2 EDTA), 300 mM NaOH, pH= 12.1) for 45 min. DMSO, Na2 EDTA
and NaOH were all obtained from Sigma-Aldrich (St Louis, MO, USA).
Electrophoresis was performed using the same solution at 250 mA and
25 V (ca. 1 V/cm) for 10 min. Unwinding and electrophoresis at
pH= 12.1 were chosen to avoid the expression of alkali labile sites,
which are extremely abundant in erythrocytes, owing to the highcondensation of chromatin (Singh et al., 1989; Tice et al., 2000; Frenzilli
et al., 2009). Our procedure thus facilitated the expression of single anddouble strand breaks, incomplete cross-links and excision repair sites(Miyamae et al., 1997; Tice et al., 2000). Electrophoresis buffer wasrinsed by dipping the slides three times in distilled water. Slides werethen immersed in 70% ethanol for 5 min, and allowed to air dry prior tostaining. Slides were stained by placing 100 µL of diluted SYBR® Gold
(Trevigen) onto each circle of dried agarose. Slides were scored under a
Nikon Eclipse 600 microscope with a Mercury lamp ultraviolet lightsource, equipped with a QImaging micropublisher 3.3 camera, using the
HCSA Comet Analysis System (Loats Associates, Inc., Westminster, MD,
USA), which automatically provides multiple standardized measures ofindividual cells, including comet tail length, percent of total cellular DNA
in the tail and tail moment. Each sample was run in duplicate on two
separate slides and 75 cells were scored blind to the collection site. Only
red blood cells were scored, while leukocytes, that are easily
recognizable because of their much bigger size, were excluded fromthis analysis.
Cytotoxicity was checked through a neutral diffusion assay(Vasquez and Tice, 1997), which determines the frequency of cellswith low molecular weight DNA. Single-cell suspensions from all
tissues were processed in the comet assay except for DNA unwinding
and electrophoresis, which were not performed (Vasquez and Tice,
1997). The nuclei were qualitatively examined for large or extended
halos as evidence of compromised cellular integrity, according to
Rider et al. (2006). There were no observable differences among thesamples, which suggested that cell viability did not differ amongradiation-level groups.
2.3. Statistical analyses
For in vivo studies, the unit of comparison in statistical analyses
assay is the individual animal (Lovell et al., 1999). For each individual
included in our analyses, we thus reduced all the measures collected
at the level of single cells to different summary statistics representing
the distribution of comet values (Lovell et al., 1999). Genotoxic effects

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273

Fig. 1. Location of the study sites. 1. Vesniane (51 18′ N, 29 37′ E), 2. Pysky (51 05′ N, 30 03′ E), 3. Dytiaku (51 07′ N, 30 09′ E), 4. Ghovtnere (50 12′ N, 30 49′ E).

detected by the comet assay are usually marked by a change in bothmean values and dispersion of the data (Duez et al., 2003). However,considerable uncertainty exists about which statistic is moremeaningful for summarizing the data collected at the level of single
cells (Lovell et al., 1999). We thus employed both the mean andmedian as two different measures of the central tendency of the
distribution. In addition, for each individual we also computed the75th percentile, this latter statistic being less sensitive to extreme
values within the distribution (Lovell et al., 1999; Duez et al., 2003).
We then tested each of these computed statistics for a relationshipbetween radiation level and DNA damage. As measures of DNA
damage, we relied on percent DNA in tail. This parameter is probably
the most commonly employed in the literature, and it has been shown
to be among the most reliable for detection of genotoxic effects andestablishment of dose–response curves (Kumaravel and Jha, 2006).
Expectations about the damage level in each site were checked bymeans of ordered heterogeneity test (Rice and Gaines, 1994). This test
employs a composite statistic (rsPc), where rs is the Spearman's rankcorrelation between the observed and expected rankings of the groups,
and Pc is the complement of the P value from the ANOVA (i.e., Pc =1 −P).
Thus, the Pc statistic extracts the information about magnitude ofvariation among the different groups and the rs statistic extracts theinformation about ordering of the groups. Pc is used instead of the P value
so that larger values entail more evidence against the null hypotheses of
equality among the different groups (Rice and Gaines, 1994). The productrsPc becomes increasingly large as the data increasingly refute the null
hypothesis in the direction of the alternative hypothesis (Rice and Gaines,
1994). This statistical test thus allows not only to test for the existence of astatistically signiﬁcant difference among groups for the variable
examined, but also to check for conformity of observed rankings of thedifferent groups to their expected rankings. We thus assigned each
population in our sample an expected ranking based on measures ofradiation level, and calculated the Spearman's rank correlation between
their observed and expected rankings. The two intermediate-level sites
were assigned the same expected ranking. The values of the composite
statistic rsPc computed for each of the three summary statistics of DNA
damage (i.e. mean, median and 75th percentile DNA in tail) werechecked for statistical signiﬁcance using the tables provided by Rice andGaines (1994).
In addition, data were analyzed by means of general linear mixed
models, with radiation level as a three-level ﬁxed effect and collection
site as a random effect. We also checked if the effect of radiation on
genetic integrity depended upon individual sex and collection date, by
including sex as a two-level factor, interaction between sex andradiation level, and sample collection date as a covariate. None ofthese factors reached statistical signiﬁcance in any of the models, andthey were thus excluded from the ﬁnal models (details not shown).
Post-hoc, pairwise tests were conducted when a statistically significant effect of radiation level was detected, and the Sidak correction
for multiple testing was applied.
3. Results
Mean, median and 75th percentile values of percent of DNA in tail(Table 1) all followed a Gaussian distribution in each of the three

Table 1
Mean values and standard errors (SE) for the mean, median and 75th percentile ofpercentage DNA in tail in the three experimental groups (i.e. low-, intermediate andhigh-level radiation). The number of individuals in each group (N) is also indicated.
Mean values represent marginal means as estimated from the model including
radiation level as a ﬁxed effect and population (i.e. collection site) as a random effect.
Parameter

Radiation

Mean

SE

N

Low
Intermediate
High

49.70
60.62
68.41

3.24
5.16
6.21

33
13
9

Low
IntermediateHigh
75th percentile tail DNA
Low
Intermediate
High

51.69
62.47
71.30

3.77
6.02
7.23

33
13
9

68.41
77.51
77.86

3.61
5.75
6.91

33
13
9

Mean tail DNA

Median tail DNA

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different groups (Kolmogorov–Smirnov test for normality, all P values
N0.05), thus meeting the requirements for parametric statistical tests(see Fig. 2 for statistical distributions of individual values in eachgroup). Individual- and population-level values are provided inTables 2 and 3.
The ordered heterogeneity test (Rice and Gaines 1994; see above)
disclosed a signiﬁcant relationship between radiation level and DNA
damage, both for mean and median percent of DNA in tail (meanpercent DNA in tail: rs = 0.949, rsPc = 0.93, P b 0.01; median percent
DNA in tail: rs = 0.949, rsPc = 0.91, P b 0.01). The increase in 75th
percentile of percent DNA in tail was not signiﬁcant (rs = − 0.632,
rsPc = − 0.46, P N 0.05).
A signiﬁcant effect of radiation level was found using generalized
linear mixed models for analyses of mean percent of DNA in tail(F2,52 = 4.26, P= 0.019; Table 1). After the Sidak correction for multiple
comparisons, this difference was found to be explained by a signiﬁcant
increase in damage from the low-dose group to the high-dose group(P= 0.030), while no signiﬁcant difference emerged between the lowdose group and the intermediate-dose group, or between the intermediate- and the high-dose group (P= 0.219 and P= 0.711, respectively).

Qualitatively similar results were obtained if individual medianvalues were used, instead of mean values, as summary statistics(F2,52 = 3.34, P = 0.043; Table 1). Percentage of DNA in tail increased
from the low- to the high-dose group, although the difference
between the two groups was marginally non-signiﬁcant after the
Sidak correction was applied (P = 0.058). The difference between the
low-dose and the intermediate-dose group, and between theintermediate- and the high-dose group was not statistically signiﬁcant (P = 0.353 and P = 0.729, respectively).
No signiﬁcant variation among radiation-level groups was found if
the 75th percentile was used for summarizing values at the level ofindividual cells (all P values N 0.05; Table 1).
In addition, no among-populations difference existed in any of theanalyses (all P values N 0.05; Table 3).
4. Discussion
After the Chernobyl accident in 1986, various studies aimed atevaluating the outcomes of the disaster have reported an increase in
mutation rate in organisms as diverse as wheat, Scots pine, oligochaetes,

Fig. 2. Frequency distribution of the mean values for percent DNA in tail in the three experimental groups. (a)–(d)–(g): frequency distribution of mean percent DNA in tail in the low-,intermediate and high-dose group; (b)–(e)–(h): frequency distribution of median percent DNA in tail in the low-, intermediate and high-dose group; (c)–(f)–(i): frequency distribution of
75th percentile of percent DNA in tail in the low-, intermediate and high-dose group.

A. Bonisoli-Alquati et al. / Comparative Biochemistry and Physiology, Part C 151 (2010) 271–277
Table 2
Mean percentage of DNA in tail for each individual bird included in the analyses.
Population (i.e. collection site), radiation level (low (L), intermediate (I) or high (H)),
and individual sex (male (M) or female (F)) are also indicated.

Table 3
Mean and standard error (SE) of mean, median and 75th percentile of percentage ofDNA for each population (i.e collection site). Number of birds from each population (N)and radiation level (low (L), intermediate (I) or high (H)) are also indicated.

Ring number

Sex

Population

Level

Mean

Statistic

1403
1406
1408
1408
1422
1423
1424
1425
1426
1430
1431
1436
1438
1441
A053382
A053388
A053393
1404
1407
1411
1413
1414
1415
1417
1421
1427
1428
1432
1433
1434
1442
A053368
A058110
A050570
A053296
A053321
A050675
A050675
A058984
A059029
A059041
A079027
A058985
A058988
A058990
A059032
B033785
B033785
B033796
A050653
A053307
B033789
B033793
B033797
B033798

F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
F
F
F
F
F
F
M
M
M
M
F
F
F
M
M
M
M
M
M

Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Ghovtnere
Dytiaku
Dytiaku
Dytiaku
Pisky
Pisky
Pisky
Pisky
Pisky
Pisky
Pisky
Pisky
Pisky
Pisky
Vesniane
Vesniane
Vesniane
Vesniane
Vesniane
Vesniane
Vesniane
Vesniane
Vesniane

L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
I
I
I
I
I
I
I
I
I
I
I
I
I
H
H
H
H
H
H
H
H
H

6.25
84.32
3.16
50.96
44.36
57.89
47.34
43.01
44.97
52.39
64.43
60.32
45.41
45.03
54.08
53.36
47.13
83.31
4.16
57.98
63.59
2.85
86.77
86.19
52.64
57.04
60.85
39.12
42.94
34.58
55.48
43.85
64.43
70.65
58.79
59.02
40.39
85.55
74.72
55.77
61.92
48.38
64.45
64.50
64.96
38.95
73.89
72.09
69.41
51.01
48.77
75.87
78.15
74.07
72.46

Mean percent DNA in tailGhovtnere
Dytiaku
Pisky
VesnianeMedian percent DNA in tailGhovtnere
Dytiaku
Pisky
Vesniane
75th percentile DNA in tailGhovtnere
Dytiaku
Pisky
Vesnianemice, birds and humans (Dubrova et al., 1996; Kovalchuk et al., 2000;
Kal'chenko and Fedotov, 2001; Weinberg et al., 2001; Tsytsugina andPolikarpov, 2003; Tsyusko et al., 2006; review in Møller and Mousseau,
2006). Moreover, the germline mutation rate was found to be elevatedin each of the four studies in which it was measured, compared tocontrol populations (Ellegren et al., 1997) or to ‘internal’ controls (e.g.
sibling conceived before the accident; Weinberg et al., 2001). However,other studies have failed to ﬁnd an increase in somatic mutations inother species (e.g. voles of the genera Microtus and Clethrionomys, and apoint mutation mouse model, the Big Blue® Transgenic Rodent
Mutation Assay System) (Wickliffe et al., 2002, 2003; Meeks et al.,2007; see also Chesser and Baker, 2006, and references therein).
Here, we report the ﬁrst ﬁnding of an increase in DNA damage
using the comet assay in any wild population of animals inhabiting275

Population

N

Level

Mean

SE

33
3
10
9

L
I
I
H

49.70
62.82
59.96
68.41

3.78
3.91
4.62
3.60

33
3
10
9

L
I
I
H

51.69
67.70
60.91
71.30

4.34
4.33
6.20
3.46

33
3
10
9

L
I
I
H

68.41
79.98
76.77
77.85

4.45
3.87
2.90
2.77

the area around Chernobyl heavily contaminated with radionuclides.
We suggest that this ﬁnding may be due to (i) the direct effect ofionizing radiation on the genetic material; or (ii) through genomic
damage caused by elevated oxidative stress. Note these causative
pathways are not mutually exclusive. In the latter case, the presenceof antioxidants as free-radical scavengers can inﬂuence the genomic
damage induced by chronic exposure to ionizing radiation.
Indeed, strand breaks (i.e. one of the genetic lesions typicallydetected by the comet assay; Tice et al., 2000) are usually regarded as
being a characteristic lesion induced by ionizing radiation (Fairbairn
et al., 1995), and it has been shown that incomplete or incorrect repairof these lesions may ultimately lead to chromosome damage and cell
death (Iliakis, 1991). On the other hand, ionizing radiation has beenshown to induce intracellular oxidative stress (Clutton et al., 1996),
leading to reactive oxygen species (ROS) formation (Halliwell andGutteridge, 2007). Active oxygen radicals can in turn efﬁciently
produce allelic deletion and permanent chromosomal instability andaberrations (Moody and Hassan, 1982; Fairbairn et al., 1995), withpossible trans-generational effects due to the transfer of damaged
genetic material to the progeny. For example, hydrogen peroxide(H2O2), a common product of a variety of oxidative stresses, has been
demonstrated to induce genetic damage leading to mutagenesis(Imlay and Linn, 1988; Imlay et al., 1988). As a result, ionizing
radiation has been proposed to induce an increase in mutation rate
through a depletion of available antioxidants, which can be utilized
for scavenging free radicals that arise from radiolysis of water andother biomolecules (Neyfakh et al., 1998a,b; Kumerova et al., 2000).
Indeed, high levels of radiation in people heavily exposed to theChernobyl fallout have been found to negatively predict individual
antioxidant defense because of their use for elimination of active
oxygen species (Neyfakh et al., 1998a,b; Kumerova et al., 2000). In acomet assay study, a signiﬁcant, negative correlation has been
observed between plasma level of carotenoids, a major class ofantioxidant compounds, and oxidative DNA damage measured asoxidized bases in lymphocytes (Collins and Horváthová, 2001).
Previous ﬁndings in our model species are also consistent with thisframework. Antioxidant levels (carotenoids, Vitamins A and E) in barnswallow blood, liver and eggs were found to be reduced in Chernobyl
compared to uncontaminated control areas (Møller et al., 2005b) andthese reductions were the best predictor of the observed increased
frequency of sperm aberrations in males from the Chernobyl region(Møller et al., 2005a). Moreover, in a recent study of this same species
where we evaluated total antioxidant capacity and the level ofmetabolites of reactive oxygen species (reactive oxygen metabolites
or ROMs), we found that ROMs were highest in the most contaminatedsites (Bonisoli-Alquati et al., in press). The ratio of the plasma level of276

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these compounds to the plasma antioxidant capacity was also increased,thus indicating that the overall balance between the antioxidant
defenses and the oxidative threat they have to face was also affected(Bonisoli-Alquati et al., in press). Moreover, an inter-speciﬁc survey ofspecies abundance in the region found that species-speciﬁc behavioral
correlates of antioxidant use (in the context of maternal allocation to theeggs, for quenching ROS generated by migratory ﬂight, and for sexualsignaling) were predictors of decline in abundance in highly contaminated sites (Møller and Mousseau, 2007b). As a whole, these ﬁndings
were thus consistent with the hypothesis of a link between radiationand individual level of antioxidants, suggesting that mutation rates may
differ within natural populations of animals owing to individual
differences in the abundance of antioxidants (Møller et al., 2005a,
2008a). We consider the present ﬁnding of a positive relationshipbetween background radiation level and genetic damage to becomprised within this previously deﬁned framework, for which it
represents an important missing link.
For results of the comet tests to be comparable among different
laboratories, it has been advised that percent of DNA in the tail in control
samples should be within 10–20% (Tice et al., 2000). Although thehigher level of damage detected in our study is outside of theserecommendations, there are a number of possible explanations,
including elevated ROS levels in migrating birds (Costantini et al.,2007). However, we are conﬁdent that the levels of DNA damage
observed in our study do not confound our overall interpretations,because all samples were handled and treated according to the same
protocol. Future studies will be designed to better understandendogenous and environmental sources of DNA damage in this species.
The biological consequences of the disaster are still debated, though
the popular description of the Chernobyl region as a thriving ecosystemappears to be based more on narrative, anecdotal evidence than onquantitative, rigorous assessment of ﬁeld procedures and data (seeMøller et al., 2008b). Indeed, some of the studies cited by Møller andMousseau (2006) are not included in the deﬁnitive (sic) survey of the
UN Chernobyl Forum (2005). Therefore, the optimistic view summarized in this report seems not rooted in state-of-the-art scientiﬁcknowledge about the possible harmful consequences of the Chernobyl
disaster.
The recent claim for a conjunct effort of the scientiﬁc community
in exploring the ecological consequences of this disaster, as well as forsetting standards for radioecological inquiries, remains largely
unanswered, despite the fact that this claim has been raised by bothsides of the controversy (e.g. Chesser and Baker, 2006; Møller andMousseau, 2006).
Moreover, little is known about the life history and evolutionary
consequences of exposure to radioactive contaminants, with some
remarkable exceptions for barn swallow and other passerine species. In
the barn swallow, a decline has been shown in reproductive
performance and output and reduced adult survival rate (Møller et al.,2005a), as well as in population sizes in the most contaminated areas(Ellegren et al., 1997; Møller et al., 2006). These ﬁndings were recently
extended to the inter-speciﬁc level, with large-scale censuses showing adecline in both abundance and richness of bird and invertebrate species
likely caused by susceptibility to the effects of exposure to radiation in
Chernobyl (Møller and Mousseau, 2007a,b, 2009a,b).
We suggest that future studies should improve study design, by
utilizing fresh blood, and extending the tests to a higher number ofindividuals and a wider geographic area, to include European populations outside Ukraine as reference populations. The test for a
relationship between DNA damage and behavioral and physiologicaltraits will also allow the assessment of ﬁtness consequences of DNA
damage in natural populations. Inference about consequences ofcontaminants on survival and reproductive success should be theprimary aim of ecotoxicological studies (Jha, 2008). In our study species,
physiological analyses of oxidative stress levels and enzyme-modiﬁed
versions of the comet test could both be employed in the future fordetecting speciﬁc damage categories (e.g. oxidative damage) andinvestigating mechanistic processes linking life-history and population-level phenomena to the underlying biochemical processes.
Acknowledgements
We are grateful to R. R. Tice for valuable suggestions for conducting
the tests. S. Woodgate, J. Fuseler, G. Frenzilli and D. Cogni greatly helpedin setting up our experimental procedure. We wish to thank R. R. Tice,
M. Parolini and three anonymous reviewers for constructive commentson the manuscript.
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