Proc. R. Soc. B (2005) 272, 247–252
doi:10.1098/rspb.2004.2914
Published online 04 February 2005

Antioxidants, radiation and mutation as revealed bysperm abnormality in barn swallows from Chernobyl

A. P. Møller1 , P. Surai2 and T. A. Mousseau3
1

Laboratoire de Parasitologie Evolutive, CNRS UMR 7103, Université Pierre et Marie Curie, Bâtiment A, 7ème étage,
7 quai Saint Bernard, Case 237, F-75252 Paris Cedex 05, France
2
Avian Science Research Centre, Scottish Agricultural College, Auchincruive, Ayr KA6 5HW, UK
3
Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
Reduced levels of antioxidants such as carotenoids and vitamins A and E can increase DNA damage causedby free radicals. Exposure to radiation has been proposed to reduce levels of antioxidants that are used for
DNA repair and this reduction may be responsible for increased levels of mutation in radioactively contaminated areas. We test this hypothesis using field measures of antioxidants in blood, liver and eggs of the barnswallow Hirundo rustica while relating these to levels of mutation as reflected by the frequency of abnormal
sperm. Antioxidant levels in blood, liver and eggs were reduced in Chernobyl, Ukraine, compared with an
uncontaminated control area, and levels of antioxidants correlated negatively with levels of background radiation. The frequency of abnormal sperm was almost an order of magnitude higher in Chernobyl than in thecontrol area and was negatively related to antioxidant levels in blood and liver. This is consistent with the
hypothesis of a direct link between radiation and individual levels of antioxidants, suggesting that levels ofmutation differ among individuals owing to individual differences in the abundance of antioxidants.
Keywords: barn swallow; carotenoids; Hirundo rustica; sperm abnormality; vitamin A; vitamin E

1. INTRODUCTION
A deficiency of antioxidants such as carotenoids and vitamins A and E can increase DNA damage caused by free
radicals (Ames 1983; Edge et al. 1997; Bast et al. 1998;
Krinsky 1998; Rice-Evans et al. 1997; Møller et al. 2000).
Radiation has been proposed to reduce levels of antioxidants that are used for DNA repair because antioxidants
are used for removing free radicals that arise owing to radiation (Ben-Amotz et al. 1998; Ivaniota et al. 1998; Neyfakh
et al. 1998a,b; Kumerova et al. 2000). It has been suggested
that this reduction in antioxidants may be responsible forincreased levels of mutation in radioactively contaminatedareas, such as those around Chernobyl in Ukraine(Dubrova et al. 1996; Ellegren et al. 1997; Kovalchuk et al.
2000; Møller & Mousseau 2001). This increase in mutations may arise from the direct effects of radiation orindirectly through the effects of a reduction in levels of antioxidants on levels of mutations. We test the hypothesis thatelevated levels of radiation are associated with reduced
levels of antioxidants thereby causing an increase in
mutation rates, using field measures of antioxidants inblood, liver and eggs of the barn swallow Hirundo rustica
while relating these to levels of mutation as reflected by thefrequency of abnormal sperm.
Antioxidants are important biochemicals that provide
organisms with protection against the damaging effects offree radicals on DNA and other molecules (review in Surai
2003). Elevated levels of radiation, as experienced byrecovery workers and other people exposed to theChernobyl accident, are associated with diminished antioxidant defence owing to the use of antioxidants forfree-radical scavenging (Ivaniota et al. 1998; Neyfakh et al.
1998a; Kumerova et al. 2000). Such effects of radiation on
antioxidant levels are reversible since supplementation
with b-carotene and vitamins A and E can reduce theeffects of the lipoperoxidative cascade among individuals
subject to radiation (Clavere et al. 1996; Ben-Amotz et al.
1998; Neyfakh et al. 1998b). Administration of subjects
with antioxidants such as vitamin E reduced the effects ofradiation on immunity (Rana & Malhotra 1995; Moriguchi
et al. 1996) and the intestine (Empey et al. 1992; Felemovicius et al. 1995). While these studies provide important
information about the links between radiation exposureand antioxidant defence, there is no study available thatlinks the effects of radiation to antioxidant defence andlevel of efficiency measured in terms of mutation rates andDNA repair at the level of individuals. We provide such apreliminary test using a free-living bird in contaminatedand control areas in Ukraine. While blood levels reflect circulating levels of antioxidants, the liver comprises the
major body storage of antioxidants in birds (Surai 2003).
Hence, we quantified both circulating antioxidants in theplasma, and stored antioxidants in the liver, to investigate
whether circulating levels or stores of antioxidants are
depressed under the influence of radiation.
Barn swallows are migratory birds that winter in subSaharan Africa and return to the breeding grounds in
Ukraine in May. Egg-laying takes place between two andfive weeks after arrival. Natal dispersal in the barn swallow
is, on average, 0.7 km in males and 2.5 km in females, while
breeding dispersal rarely goes beyond a breeding site
(Cramp 1988; Møller 1994). Thus, dispersal between our
main study sites around Chernobyl and those in the control
area at Kanev ca. 120 km to the southeast is unlikely toaffect the results presented here. Previous studies on the

Author for correspondence (amoller@snv.jussieu.fr).

Received 11 February 2004
Accepted 18 August 2004247

# 2005 The Royal Society248 A. P. Møller and others Antioxidants and effects of radiation

1999). In addition, 87 out of the 88 partial albinos caused
by mutation had partly albinistic feathers within the redmelanin-based plumage, whereas only nine were expected,
given the area of the red plumage badge (Møller & Mousseau 2001), suggesting that partial albinism is directly
related to this melanin-based coloration. Adult barn swallow males with long tails tend to have a more strongly
reflecting red throat plumage colour than do short-tailed
males, and such birds also have higher levels of circulating
carotenoids in the blood (Saino et al. 1999). Studies ofthree microsatellites in barn swallows from Chernobyl haverevealed that mutation rates are increased by a factor of two
to ten when compared with individuals from two uncontaminated control areas (Ellegren et al. 1997).

(a)concentration (µg g–1)

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2. MATERIAL AND METHODS(a) Study area and population

15

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retinol0α-tocopherol

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antioxidant

Figure 1. Concentration of antioxidants (mg g1) in (a) bloodand (b) liver of adult barn swallows and in (c) eggs of barnswallows from Chernobyl (grey bars) and Kanev (open bars;
uncontaminated control area). Values are means (+1 s.e.).
One-way ANOVAs revealed significant differences for blood((a) F ¼ 18:58; d:f : ¼ 1; 39; p < 0:001; F ¼ 16:64; d:f : ¼
1; 39; p ¼ 0:0002; F ¼ 16:64; d:f: ¼ 1; 39; p < 0:001,
respectively); for liver retinol, a-tocopherol and carotenoids((b) F ¼ 90:70; d:f : ¼ 1; 56; p ¼ 0:001; F ¼ 22:33; d:f : ¼
1; 56; p < 0001; F ¼ 30:29; d:f : ¼ 1; 56; p < 0:001,
respectively); and for egg a-tocopherol and carotenoids((c) F ¼ 9:23; d:f : ¼ 1; 43; p ¼ 0:004; F ¼ 5:31; d:f : ¼
1; 43; p ¼ 0:03, respectively), while the difference for egg
retinol was not significant (F ¼ 1:29; d:f : ¼ 1; 43; p ¼ 0:26).

We studied barn swallows in Ukraine just outside the exclusionzone of the Chernobyl area, by visiting villages and checking collective farms for their presence. Once such a farm had been located, we recorded radiation levels and captured adult barnswallows for measurements and subsequent individual ringing.
Our own field measurements of radiation at the ground level usinga hand-held Inspector dosimeter (Model: Inspector, SE International, Inc., Summertown, TN, USA) revealed levels of mainly
c radiation of 0.390 mR h1 (s:e: ¼ 0:317) at 14 breeding sites in
the Chernobyl region. As a control area, we used Kanev ca.
120 km southwest of Kiev that has a relatively low level of contamination. Mean levels of radiation were 0.025 mR h1(s:e: ¼ 0:002) at five breeding sites. Our data were validated withcorrelation against data from the governmental measurements
published by Shestopalov (1996), estimated as the midpoint of theranges from this published source. This analysis revealed a verystrong positive relationship (linear regression on log–log transformed data: F ¼ 159:46; d:f : ¼ 1; 18; r 2 ¼ 0:89; p < 0:0001,
slope ðs:e:Þ ¼ 1:28 (0.10)), suggesting that our field estimates ofradiation were comparable to other estimates. We studied barnswallows in these two study areas during 6–12 June 2000 and 4–11
June 2002.

(b) Field procedures
We captured barn swallows with mist nets across open doors andwindows in farm buildings. This method is highly efficient during
the main breeding season since mark–recapture studies haveshown a capture probability of 98% of all individuals with just a
few capture sessions (Møller & Szép 2002). During the field season 2000, we collected blood samples of 70 ml in capillarity tubes
from a sample of 41 individuals of both sexes (21 from Chernobyl
and 20 from Kanev), and these samples were later used for quantification of circulating antioxidants in blood (x 2c). A sample of 58
birds (of which 36 were males) (29 from Chernobyl and 29 from
Kanev) was collected with permission from the National Museumof Natural History, Kiev, Ukraine, in 2000 and these individuals
were used for quantification of antioxidants in the liver and abnormality of sperm. During the field season 2002, we collected one
egg from each of a sample of 45 nests (23 from Chernobyl and 22
from Kanev) for quantifying antioxidants in eggs.

(c) Laboratory analyses of antioxidantsbarn swallow in the Ukraine have found that birds in areas
with elevated radiation around Chernobyl have more palemelanin-based plumage pigmentation than in control
areas, in particular long-tailed males (Camplani et al.
Proc. R. Soc. B (2005)

Antioxidants in plasma, liver and egg yolk were assessed by
HPLC-based methods described earlier (Surai et al. 2001a;
Blount et al. 2003). In brief, 15–30 ml of plasma was vortexed withan equal volume of 5% sodium chloride, plus two volumes ofAntioxidants and effects of radiation

75
retinol (µg g–1)

249

evaporated to dryness under nitrogen gas, then redissolved in
300 ml of a methanol–dichloromethane mixture (1 : 1, v/v).
The antioxidants were extracted from the egg yolk and liver asfollows. Egg yolk (100–200 mg) or tissue (80–100 mg) were
homogenized in 1 ml of 1 : 1 (v/v) mixture of 5% NaCl solution
and ethanol followed by the addition of 2 ml of hexane and further
homogenization for 1 min. After centrifugation, the hexane layer
was collected and the extraction was repeated twice. Hexane
extracts were combined, evaporated to dryness under nitrogengas, then redissolved in 300 ml of a methanol–dichloromethane
mixture (1 : 1, v/v).
Total carotenoids were determined as follows. Samples (10 ml)
were injected into an HPLC system fitted with a 5 m C18 reversephase column (25 cm  4.6 mm) (Spherisorb-type S5NH2; Phase
Separations, Clwyd, UK) with a mobile phase of methanol–water(97 : 3 v/v) at a flow rate of 1.5 ml min1. Carotenoids were identified as a single peak at 445 nm, and concentrations were determined using lutein as a standard (Sigma-Aldrich, Poole, UK).
Previously, using zebra finch plasma (Blount et al. 2003) we
showed that carotenoid measurements were highly repeatable(one-way ANOVA, F ¼ 3:77; d:f : ¼ 22; 26; p ¼ 0:001; interclasscorrelation coefficient, r ¼ 0:57). Similar results were also
obtained when testing a-tocopherol and retinol determinations.
The same HPLC system fitted with a Spherisorb type S3ODS2,
3 m C18 reverse phase column, 15 cm  4.6 mm was used (Phase
Separation Ltd, UK). Chromatography was performed using thesame mobile phase at a flow rate of 1.05 ml min1 and retinol anda-tocopherol were determined using fluorescent detection. Tocol
was used as an internal standard, and standard solutions of a-tocopherol and retinol (Sigma-Aldrich, Poole, UK) were used for
instrument calibration.
All analyses were conducted blind with respect to the origin ofbirds.

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α-tocopherol (µg g–1)

A. P. Møller and others

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carotenoids (µg g–1)(d) Assessing sperm abnormality
A sample of sperm was obtained from the cloacal protuberance ofall 36 males, using samples that were frozen after collection in thefield. The frequency of abnormal sperm was quantified with amicroscope at magnification 400, as the proportion of sperm
among 100 spermatozoa that had clearly abnormal morphologies(bent or missing flagella, abnormal head shapes, two heads, twotails, coiled tails, elongated heads, etc.; Gomes 1977). This measure predicts fertilizing ability in chicken and turkey (Lorenz
1969; Bakst & Cecil 1997). All analyses were done blind withrespect to the identity and the origin of birds.

15

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0.01

0.1
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Figure 2. Antioxidants in liver of adult barn swallows from
Ukraine in relation to levels of radiation (mR h1). The
regressions were significant when radiation level was logtransformed ((a) retinol (mg g1): F ¼ 22:01; d:f : ¼ 1; 56;
r 2 ¼ 0:28; p < 0:0001, slope ðs:e:Þ ¼ 21:47 (4.58);(b) a-tocopherol (mg g1): F ¼ 11:84; d:f : ¼ 1; 56;
r 2 ¼ 0:17; p ¼ 0:001, slope ðs:e:Þ ¼ 7:00 (2.04); (c)carotenoids (mg g1): F ¼ 9:51; d:f : ¼ 1; 56; r 2 ¼ 0:15;
p ¼ 0:003; slope ðs:e:Þ ¼ 2:46 ð0:80Þ, respectively).

ethanol, 500 ml hexane was then added and vortexed. Aftercentrifugation the hexane phase, containing antioxidants (carotenoids, a-tocopherol and retinol) was collected and extraction
was repeated. The resulting hexane extract was combined andProc. R. Soc. B (2005)

3. RESULTS
We investigated levels of three common antioxidants inblood and liver of barn swallows from contaminated andcontrol areas in Ukraine. There were weak but significant
positive correlations between concentrations in plasma
and in liver (log-transformed data: total carotenoids:
Pearson r ¼ 0:39; t ¼ 2:64; d:f : ¼ 39; p ¼ 0:01; retinol :r ¼ 0:38; t ¼ 2:59; d:f : ¼ 39; p ¼ 0:01; a-tocopherol : r ¼
0:36; t ¼ 2:41; d:f : ¼ 39; p ¼ 0:01). Levels of antioxidants
in blood and liver differed significantly between Chernobyl
and the control area, with concentrations in Chernobyl, on
average, being only a half to a quarter of that in the control
area (figure 1a,b). There was a significant sex effect on liverconcentration of total carotenoids, when entering sex as anadditional factor in a two-way ANOVA (analysis based on
log-transformed concentration: F ¼ 7:56; d:f : ¼ 1; 48; p ¼
0:008; mean (s.e.), males: 0.70 mg g1 (0.06), n ¼ 32;

250 A. P. Møller and others Antioxidants and effects of radiation

(c)

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abnormal sperm (%)

abnormal sperm (%)

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abnormal sperm (%)

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Figure 3. Frequency of abnormal sperm (%) in barn swallows from Ukraine in relation to radiation and liver antioxidants in adultmales. The regressions were significant when radiation level was log-transformed ((a) radiation (mR h1); F ¼ 34:07; d:f : ¼
1; 34; r 2 ¼ 0:50; p < 0:0001; slope ðs:e:Þ ¼ 0:21 ð0:04Þ; (b) retinol (mg g1): F ¼ 35:98; d:f : ¼ 1; 34; r 2 ¼ 0:51; p < 0:0001;
slope ðs:e:Þ ¼ 0:005 ð0:001Þ; (c) a-tocopherol (mg g1): F ¼ 71:86; d:f : ¼ 1; 34; r 2 ¼ 0:68; p < 0:0001; slope ðs:e:Þ ¼
0:014 ð0:002Þ; (d ) carotenoids (mg g1): F ¼ 29:43; d:f : ¼ 1; 34; r 2 ¼ 0:46; p < 0:0001; slope ðs:e:Þ ¼ 0:03 ð0:01Þ,
respectively). Similar results were found for antioxidants in blood (linear regressions with radiation level log-transformed: retinol(mg g1): F ¼ 12:48; d:f : ¼ 1; 26; r 2 ¼ 0:32; p ¼ 0:002; slope ðs:e:Þ ¼ 0:74 ð0:21Þ; (c) a-tocopherol (mg g1): F ¼ 11:33;
d:f : ¼ 1; 26; r 2 ¼ 0:25; p ¼ 0:006; slope ðs:e:Þ ¼ 0:07 ð0:02Þ; (d ) carotenoids (mg g1): F ¼ 8:78; d:f : ¼ 1; 26; r 2 ¼ 0:30;
p ¼ 0:002; slope ðs:e:Þ ¼ 1:55 ð0:46Þ, respectively).

females: 0.56 mg g1 (0.07), n ¼ 20). The sex effect was not
significant for the other liver antioxidants or for blood antioxidants, and the area by sex interaction was not significant
in any of the six analyses. Therefore, we did not enter sex asa factor in the subsequent analyses. Antioxidants weremore abundant in areas with lower levels of radiation, but
decreased dramatically in the Chernobyl region (figure 2).
The concentration of a-tocopherol and total carotenoids
in eggs was reduced by 30–60% in eggs from Chernobyl
compared with eggs from the control area (figure 1c). Thissuggests that offspring developed as embryos with initiallyextremely low levels of antioxidants in the Chernobyl
region compared with the control area.
In the barn swallow, the frequency of sperm with abnormal morphology differed significantly between Chernobyl
and the control area (one-way ANOVA based on squareroot-arcsine-transformed data: F ¼ 81:99; d:f : ¼ 1; 34;
Proc. R. Soc. B (2005)

p < 0:001; mean (s.e.) for Chernobyl: 21.74% (2.41),
n ¼ 19; control: 2.65% (0.64), n ¼ 17). Abnormal sperm
were more common among barn swallows from areas withhigh levels of radiation (figure 3a), and when levels ofantioxidants in liver were low (figure 3b–d ). Similar results
were found for antioxidants in blood (figure 3). This provides evidence of individual levels of mutation, as reflectedby the frequency of abnormal sperm, increasing in situations where the background level of radiation is high, andwhen the concentration of antioxidants is low.
4. DISCUSSION
Exposure to radiation has been proposed to reduce levels ofantioxidant defence owing to their use for free-radical scavenging (Ivaniota et al. 1998; Neyfakh et al. 1998a; Kumerova
et al. 2000). However, inflammatory responses could also
cause decreased levels of antioxidants in tissues of irradiatedAntioxidants and effects of radiation

birds (Lorimore et al. 2003; Morgan 2003). We have shown,
for a free-living migratory passerine bird, that levels of carotenoids and vitamins A and E differ significantly between
populations breeding in the contaminated area around Chernobyl and a control area (figure 1). Levels were depressed in
the Chernobyl region by more than 50% compared withindividuals from the control area. There was no significant
sex effect, with the exception of plasma carotenoids, whichwere less abundant in females than in males. This difference
may relate to adult females depositing carotenoids into eggs
at this time of the year, although this explanation does not
explain the absence of a sex difference for plasma levels ofvitamins A and E or liver antioxidants that are also deposited
into eggs. These effects of radiation on antioxidant levels
were prominent in blood, liver and eggs, suggesting that both
circulating levels and storage of antioxidants differed consistently between these regions. These differences between
regions may be open to alternative explanations such as differences in availability of antioxidants in the diet. However,we were able to show that levels of antioxidants in individual
barn swallows were inversely related to levels of backgroundradiation (figure 2). This suggests that radiation (or a correlated factor) affected antioxidant levels in barn swallows. The
exact physiological mechanism responsible for this effectremains to be determined.
Birds’ eggs contain large amounts of antioxidants thatare used for free-radical scavenging by the developing
embryo (review in Surai 2003). We detected dramaticallyreduced levels of some antioxidants in eggs of barn swallows from the Chernobyl region compared with controls(figure 1c). This suggests that offspring developed with lowlevels of maternally derived antioxidants in the Chernobyl
region. There was a reduction in egg concentration of antioxidants with increasing levels of radiation (figure 2). However, there was considerable variation among sites in therange of antioxidant concentrations recorded (figure 2),
and some of this variation may be accounted for by the patchy distribution of high levels of radiation in the Chernobyl
region, even on a micro-geographical scale (Shestopalov1996). Such depression of dietary carotenoids deposited
into yolk can increase the susceptibility of embryonictissues to free-radical attack (Surai & Speake 1998), andreduce hatchling immune function (McWhinney et al.
1989; Haq et al. 1996). Avian embryos and hatchlings are
particularly in need of antioxidants because their rapid
metabolism incurs high rates of free-radical production,and their tissues are rich in unsaturated lipids that are particularly susceptible to free-radical attack (Surai et al.
2001b). We suggest that such low levels of antioxidants
may have important consequences for offspring whenreaching adulthood.
Abnormal sperm with aberrant morphology occur at a
low frequency in birds and other vertebrates (5–10% inbirds, Bakst & Cecil (1997); 10–40% in mammals, Gomes(1977)). High frequencies of abnormal sperm are typical ofmutants (Yu et al. 2000; Lamitina & L’Hernault 2002;
Mendoza-Lujambo et al. 2002), although other factors may
also contribute to the presence of abnormal sperm (Bakst &Cecil 1997). We found low levels of sperm abnormality
among barn swallows from the control area, but elevatedlevels around Chernobyl. Since levels of sperm abnormality
were positively related to levels of radiation (figure 3a), but
negatively related to levels of antioxidants in blood andProc. R. Soc. B (2005)

A. P. Møller and others

251

liver of adult barn swallows (figure 3), we can conclude thatradiation (or a factor associated with radiation) increases
levels of sperm abnormality.
Mutations are novel genetic variants, and they are supposed to occur at a constant low rate (Lynch et al. 1999).
Individual differences in mutation rates can arise from individual differences in the ability to cope with mutagens andperform DNA repair. This study has shown that levels ofantioxidants differ among individuals in relation to levels ofradiation owing to the nuclear accident at Chernobyl.
These individual differences in levels of antioxidants are
related to levels of mutation, as reflected by sperm abnormality, providing a hypothetical mechanism that links individual levels of antioxidants through exposure to radiation
to levels of mutations. This hypothesis is testable by investigating the relationship between rates of mutation in molecular markers obtained from sperm and circulating levelsof antioxidants in males that produced these sperm.
The authors are grateful to G. Milinevski, A. M. Peklo and E.
Pysanets for logistic help provided during our visits to
Ukraine. The authors received funding from the CNRS(France), the University of South Carolina School of theEnvironment, the Samuel Freeman Charitable Trust, theNational Science Foundation and National Geographic
Society to conduct this research.

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