148
Seasonal Variation in the Regulation of Redox State and Some
Biotransformation Enzyme Activities in the Barn
Swallow (Hirundo rustica L.)
Sari Raja-aho1,*,†
Mirella Kanerva1,*
Tapio Eeva1
Esa Lehikoinen1
Petri Suorsa1
Kai Gao1
Dalene Vosloo2
Mikko Nikinmaa1
1
Department of Biology, University of Turku, 20014 Turku,
Finland; 2School of Biological and Conservation Sciences,
University of KwaZulu-Natal, Bag X54001, Durban 4000,
South Africa
Accepted 1/21/2012; Electronically Published 3/7/2012
ABSTRACT
Little is known of the normal seasonal variation in redox state
and biotransformation activities in birds. In long-distance migratory birds, in particular, seasonal changes could be expected
to occur because of the demands of migration and reproduction. In this study, we measured several redox parameters in
the barn swallow (Hirundo rustica L.) during the annual cycle.
We captured the wintering barn swallows before spring migration in South Africa, and we captured the barn swallows that
arrived in spring, bred in summer, and migrated in autumn in
Finland. The redox status and biotransformation activities of
barn swallows varied seasonally. Wintering birds in South Africa
had high biotransformation activities and appeared to experience oxidative stress, whereas in spring and summer, they
showed relatively low redox (superoxide dismutase [SOD], catalase [CAT], and glutathione reductase [GR]) and biotransformation enzyme activities. Autumn birds had very low biotransformation enzyme activities and low indication of
oxidative stress but high activity of some redox enzymes (GR
and glucose 6-phosphate dehydrogenase [G6PDH]). High activities of some redox enzymes (SOD, GR, and G6PDH) seem
to be related to migration, whereas low activities of some redox
enzymes (SOD, CAT, and GR) may be associated with breeding.
Barn swallows in South Africa may experience pollution-related
* S.R. and M.K. contributed equally to this article.
† Corresponding author; e-mail: [email protected].
Physiological and Biochemical Zoology 85(2):148–158. 2012. 䉷 2012 by The
University of Chicago. All rights reserved. 1522-2152/2012/8502-1130$15.00.
DOI: 10.1086/664826
oxidative stress, which may hamper interpretation of normal
seasonal variation in redox parameters.
Introduction
Environmental monitoring using birds often includes biomarkers related to redox state, which is determined by the
balance between oxidants and antioxidants (Beckman and
Ames 1998; Finkel and Holbrook 2000; Halliwell and Gutteridge 2007). If an organism is experiencing oxidative stress (i.e.,
if the redox state is shifted to the oxidative direction), the
formation of various reactive oxygen species (ROS) exceeds the
organism’s antioxidant defense capacity, first causing disturbances in cellular signaling dependent on ROS and, if more
severe, directly damaging biomolecules, such as DNA, proteins,
and lipids (Beckman and Ames 1998; Finkel and Holbrook
2000; Valavanidis et al. 2006; Halliwell and Gutteridge 2007;
Monaghan et al. 2009). The net effect of oxidative stress may
be seen in breeding success, life span, activity, and many functional responses, such as the immune response (Costantini
2008). Because oxidative stress is often associated with increased
energy turnover, seasonal variation might occur, especially in
long-distance migrating birds (McWilliams et al. 2004; Costantini et al. 2007). To assess the oxidative stress caused by
chemical exposure, one needs to understand the normal seasonal changes in redox parameters, including relevant enzyme
activities (Stohs and Bagchi 1995).
The enzymes involved in regulating redox balance include
glutathione S-transferase (GST), glutathione peroxidase (GP),
glutathione reductase (GR), superoxide dismutase (SOD), and
catalase (CAT; Halliwell and Gutteridge 2007; Costantini 2008;
Monaghan et al. 2009; Koivula and Eeva 2010). Small antioxidant molecules involved in the regulation of redox balance
can be obtained via food, such as carotenoids, flavonoids, and
vitamins E and C, or can be produced endogenously, such as
glutathione (GSH), which is the major small redox state–regulating molecule (Halliwell and Gutteridge 2007; Costantini
2008; Monaghan et al. 2009). Additional important molecules
involved in the regulation of redox state are thioredoxins (Nishiyama et al. 2001), although their presence and induction in
birds as a result of redox disturbances have not been studied.
The biomarkers used in studies of oxidative stress in birds are
discussed in detail in a recent review by Koivula and Eeva
(2010). Oxidative stress is often associated with changes in
Seasonal Variation in Redox State and Biotransformation Activity in Birds
149
Table 1: Data on free-living barn swallows captured from roosts in South Africa and Finland during the
annual cycle in 2007
Season
No. barn swallows
Male sex
Female sex
Capture dates
Capture place
Winter
Spring
Summer
Autumn
25
24
22
23
15
22
22
16
10
2
0
7
Feb. 21–Mar. 3
May 21–23
June 26, July 5–16
Aug. 21–Sept. 19
Potchefstroom, South Africa
Petteby, Finland
Petteby, Finland
Petteby, Finland
Total
94
75
19
biotransformation activity (Halliwell and Gutteridge 2007).
Compounds that activate the aryl hydrocarbon receptor (AhR)
and consecutive xenobiotically induced gene expression, including biotransformation enzymes of the cytocrome P450
family, cause the formation of oxygen free radicals (Whyte et
al. 2000). Activation of AhR is often determined using ethoxyresorufin-O-deethylase (EROD) enzyme activity (Lorenzen et
al. 1997).
One shortcoming in the use of redox state biomarkers and
biotransformation enzyme activities in environmental studies
is the general lack of the knowledge regarding their seasonal
variation. Knowledge regarding the function of different antioxidants, their levels in the blood or tissues of birds, and the
seasonal variation in antioxidant activity is still scarce. Only a
few studies have monitored the changes throughout the whole
year (Norte et al. 2008a, 2008b, 2009; Cohen et al. 2009). The
source of variation in the measured parameters is often difficult
to ascertain for adult birds because of their unknown exposure
to xenobiotics. The parameters used in biomonitoring may also
change within the different stages of the life cycle of birds. For
example, marked changes occur during nestling development
in some hole-nesting insectivorous passerines (Tanhuanpää et
al. 1999). Seasonal physiological processes, such as molt (Franson et al. 2002), reproduction (Alonso-Alvarez et al. 2004;
Wiersma et al. 2004; Bertrand et al. 2006), and heavy exercise
during migration, cause variation in the redox state of wild and
captive birds (Costantini et al. 2007, 2008; Larcombe et al.
2008). Because information on these changes is scarce, we studied the seasonal variation in parameters commonly used to
describe redox state and biotransformation activity using a
long-distance migratory bird, the barn swallow (Hirundo rustica
L.), as a model species.
Material and Methods
Study Area and Data
The free-living adult barn swallows (n p 94 ) used in this study
were captured from roosts during four different seasons in 2007
(table 1). Barn swallow populations breeding in Sweden, Finland, the Baltic countries, or farther east most likely winter in
the eastern parts of South Africa, such as the Johannesburg
area (Szep et al. 2006, 2007; Ambrosini et al. 2009). Ringing
recoveries of the Finnish barn swallows in eastern South Africa
also verify this; altogether, 38 (49%) of 78 Finnish barn swallows
recovered in Africa were recovered in eastern South Africa, and
11 of these were recovered from the area close to Johannesburg
(data obtained from the Finnish Ringing Centre). We captured
the wintering barn swallows before spring migration in Potchefstroom, South Africa (26⬚42S, 27⬚06E; ∼100 km from Johannesburg). Barn swallows that were arriving in spring, breeding in summer, and migrating in autumn were captured from
the common roosts in Petteby, Finland (60⬚17N, 22⬚11E). We
chose the dates of seasonal captures carefully on the basis of
the well-documented phenology of Finnish barn swallows
(Finnish Ringing Centre). Because we did not capture birds
from the nests, we could not determine the exact breeding status
of summer birds. On the basis of testis size (measured in mm),
we can assume that the barn swallows were in a breeding phase.
Most barn swallows arrive in Finland in the first or second
week of May and lay their eggs (first brood) in the beginning
of June (in 2007, laying started in 85 of 404 nests before May
24; calculated according to Saino et al. 2004). The mean hatching day of the first brood was June 19 (data not shown). Barn
swallows breeding the second time usually start during the end
of June (in 2007, the mean hatching day of the second brood
was July 29; data not shown). By the end of August, most adult
barn swallows are ready for migration, and no visual signs of
breeding (e.g., incubation patch and cloacal protuberance) can
be found.
We measured the individuals for maximum wing length
(Svensson 1992), longest and shortest tail feathers, fat score
(ranging from 0 [no subcutaneous fat] to 8 [fat layer covers
the ventral side completely]; Kaiser 1993), and body mass (measured to the nearest 0.1 g with a spring balance). In addition
to determining the age of the barn swallows, we determined
their sex (75 males and 19 females) according to Jenni and
Winkler (1994) and Svensson (1992). We then killed the barn
swallows by cervical dislocation and pressed the trachea with
the thumb to make sure of a quick death. Barn swallow carcasses were dissected, and liver samples were collected and immediately frozen in liquid nitrogen. The remainder of each
carcass was frozen (initially at ⫺5⬚C; carcasses were subsequently maintained at ⫺20⬚C) for further analyses. Sex of the
barn swallows was confirmed by examining the gonads. We
used residual mass (deviations from linear regression of body
mass on wing length) and fat score as measures of energetic
condition in the following analyses. The residual mass also takes
into account nonlipid energy sources, such as muscles, and is
150 Raja-aho, Kanerva, Eeva, Lehikoinen, Suorsa, Gao, Vosloo, and Nikinmaa
therefore a more general measure of condition than is fat score
(Long and Holberton 2004).
Barn swallows molt completely in the winter quarters. After
the molt of tail feathers in Africa, determining the age of barn
swallows on the basis of plumage characteristics is impossible.
Because their molt is slow (duration, 121–185 d), compared
with that of most small passerines, barn swallows may start the
spring migration while still molting (Jenni and Winkler 1994).
We checked the molt stage in wing (primaries and secondaries)
and tail feathers of 25 African barn swallows. We used the
primary score of wintering individuals (ranging from 0 for old
feather to 5 for new feather, summed over nine developed
primaries and thus ranging from 0 to 45; Ginn and Melville
1983). With the help of the primary score and the known
minimum duration of the molt (121 d; Ginn and Melville
1983), we calculated the minimum number of days each individual still needed to finish the molt (completion of molt p
(45 ⫺ primary score)/(45/121)). Duration of primary molt can
be used as a proxy for total molt duration, because it usually
extends over the entire molt period (Jenni and Winkler 1994).
Because the molting of primaries at the tip of the wing was
incomplete, we needed to estimate the final wing length of
wintering barn swallows to calculate the size-independent residual mass used in the analyses. The final wing length was
estimated on the basis of fully grown wing feathers of spring
barn swallows (n p 24). First, we measured the wing length
up to the newly molted, already complete primaries (fifth
through eighth). Then, using the mean values of length differences between each of the primaries of spring barn swallows,
we estimated the missing length of the molting wing and added
the estimate to the measured length to obtain the final wing
length.
The experiments that we performed comply with the current
laws of Finland and South Africa. The licences for animal experiments were granted by the Southwest Finland Regional
Environment Centre (LOS-2006-L-416-254 and LOS-2007-L22-254), the Central Animal Laboratory of the University of
Turku (1661/06), and the South African Department of Agriculture, Conservation, Environment and Tourism (000377NW06 and 000054NW-07).
Sample Processing
Frozen liver pieces were crushed in liquid nitrogen with a mortar and pestle. Thereafter, a piece of liver (approximately
0.5 # 0.5 cm) was homogenized in 600 mL of cold 0.1 M
K2HPO4 plus 0.15 M KCl buffer (pH 7.4) using TissueLyser
(Qiagen), 30 s⫺1. A 100-mL portion of the homogenate was
pipetted into an Eppendorf tube that contained 10 mL of 33
mM 1-methyl-2-vinylpyridinium-trifluoromethanesulfonate
(Sigma Chemicals) in 0.1 M HCl, for the determination of
oxidized glutathione (GSSG). A 50-mL proportion of homogenate was pipetted in an Eppendorf tube for the determination
of reduced GSH level. Both tubes were frozen immediately in
liquid nitrogen and stored at ⫺80⬚C until measurements were
determined. The rest of the homogenate was centrifuged for
15 min at 10,000 g (⫹4⬚C). The supernatant was divided into
aliquots, frozen in liquid nitrogen, and stored at ⫺80⬚C.
Determination of Enzyme Activities and Small-Molecule Levels
The parameters were measured in triplicate (intra-assay coefficient of variability [CV] !10% in all cases) using 96-well and
384-well microplates, which in most cases required reducing
reagent volumes, compared with volumes recommended in the
method instructions. Because we used three control samples in
every plate, we were able to correct the interassay precision
(CV, 4%–35%) with the ratio specific to the particular plate
(range, 0.8–1.2). The levels of GR, GP, GST, and CAT activity
were measured with Sigma kits (Sigma Chemicals). CAT activity
of summer birds was below the detection limit of the method
and therefore could not be measured. The GP activity was
measured using 2 mM H2O2 as the substrate. The inhibition
rate of SOD was measured using a Fluka kit (Fluka). The ratio
between reduced and oxidized glutathione (GSH/GSSG ratio)
and the total glutathione content (including both the reduced
and oxidized forms of glutathione; totGSH) were measured
using an OxisResearch kit (OxisResearch). Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured according to Noltmann et al. (1961), and the EROD activity was
measured according to Burke and Mayer (1974). Lipid hydroperoxides (LHPs) were measured using the FOX2 method,
modified from Nourooz-Zadeh et al. (1995) and Bou et al.
(2008), using cumene hydroperoxide as a standard (Sigma
Chemicals). The protein content was determined with the Bradford method (Bradford 1976) using BioRad protein assay reagent (BioRad) with bovine serum albumin (Sigma Chemicals)
as a standard. All of the measurements were performed with
an Envision plate reader (Perkin-Elmer) except for protein content determinations, which were performed with a Victor 1
plate reader (Perkin-Elmer).
Statistical Methods
We analyzed all data using generalized linear models (GLMs)
with the GLIMMIX procedure of SAS, version 9.2 (SAS 2008)
and principal component analysis (PCA) with Predictive Analytics Software statistics 18.0. Response variables GST and GR
distributed normally (checked from the model residuals). For
the nonnormally distributed data we used lognormal distribution (EROD, totGSH, GSH/GSSG ratio, LHP, SOD, CAT, GP,
and G6PDH). We calculated 95% confidence intervals (CIs) to
establish bounds for the true effect (Steidl and Thomas 2001).
First, we used GLMs to analyze the seasonal variation in
enzyme activity of EROD, GST, totGSH, GSH/GSSG ratio, LHP,
SOD, CAT, GP, GR, G6PDH, and protein content. Pairwise
comparisons adjusted for the number of tests were made from
estimated marginal means (EMMs) with the Tukey-Kramer test.
Second, we used PCA to discriminate the patterns of variation
in the measured variables. PCA was performed using all individual enzyme activities (except for CAT) and other molecule
levels, residual mass, and fat score as input variables (no ro-
Seasonal Variation in Redox State and Biotransformation Activity in Birds
tation was used). The requirements for PCA were tested with
the Kaiser-Mayer-Olkin (KMO) test and Bartlett’s test for sphericity (KMO 1 0.5; P ! 0.05, by Bartlett’s test). We also viewed
the correlation coefficients, significance levels, and communalities to detect any nonsignificant parameter correlations
(r ! 0.3, P 1 0.05) or low communalities (h 2 ! 0.3). Finally, we
tested the relationship between different body condition variables and the biomarkers separately for each season. Sex, residual mass, fat score, gonad size (mm), and completion of
molt (for wintering birds only) were used as explanatory variables in the models. Because spring and summer birds were
mainly males, and because sex was not associated with the
measured parameters in winter or autumn birds (only the GR
activity in the autumn group differed between females and
males; 5.72 mmol⫺1 min⫺1 mg vs. 4.93 mmol⫺1 min⫺1 mg), we
did not separate sexes in any of the subsequent analyses.
Results
All the measured biotransformation and redox state parameters
showed statistical differences among seasons (table 2; figs. 1,
2). Biotransformation activity, as given by EROD and GST
activities, is elevated among wintering barn swallows, compared
with other groups (fig. 1A, 1B). An increase in the totGSH and
a decrease in the GSH/GSSG ratio in wintering birds indicate
that they are experiencing oxidative stress (fig. 1C, 1D). These
birds also showed a tendency toward having the highest LHP
levels (fig. 1E), indicating the damage done by ROS, although
LHP levels in the winter group were not statistically significantly
different from those in the spring and summer groups. The
SOD inhibition rates and CAT activities were highest in the
winter group and lower in the spring (for SOD inhibition rate
and CAT activity) and summer (for SOD inhibition rate only)
groups (fig. 2A, 2B). GP activity was lowest in the autumn birds
(fig. 2C), which possibly explains the high GSH/GSSG ratio
among those birds (fig. 1D). The levels of GR and G6PDH
activity were elevated in winter and autumn birds (fig. 2D, 2E).
Three components showing the eigenvalue 11 together accounted for 57% of the variation (table 3). Component 1 (PC1)
showed positive loadings by the parameters totGSH, GST,
EROD, LHP, and SOD and negative loadings with residual
mass. Component 2 (PC2) was positively related to the parameters G6PDH, GR, and GSH/GSSG ratio and was negatively
related to parameter GP. Fat score was positively related to
component 3 (PC3; table 3). The first two principal components (PC1 and PC2) together indicate increased biotransformation activity and oxidative stress in winter quarters, reduced
biotransformation activity but intermediate oxidative stress
during the breeding period, and reduced biotransformation
activity and oxidative stress in autumn (fig. 3).
The residual mass of spring barn swallows was negatively
associated with LHP and GR (table 4). Residual mass of wintering birds was positively associated with G6PDH (table 4).
The fat score of barn swallows in summer was positively associated with GR and G6PDH (table 4). The size of male testis
(mm) in summer was positively associated with totGSH and
151
Table 2: Generalized linear model statistics for the seasonal
variation in biotransformation activity and redox state of
barn swallow during the annual cycle
Dependent variable
a
EROD
GSTb
totGSHa
GSH/GSSG ratioa
LHPa
SODa
CATa
GPa
GRb
G6PDHa
F
14.60
43.26
25.69
93.03
4.57
3.19
4.55
3.31
9.97
9.19
df
3,
3,
3,
3,
3,
3,
2,
3,
3,
3,
90
90
90
90
90
90
53c
90
90
90
P
!.0001
!.0001
!.0001
!.0001
.0005
.0275
.0150
.0238
!.0001
!.0001
Note. CAT p catalase; EROD p ethoxyresorufin-O-deethylase; GP p
glutathione peroxidase; GR p glutathione reductase; GSH p reduced
glutathione; GSSG p oxidized glutathione; GST p glutathione S-transferase;
G6PDH p glucose-6-phosphate dehydrogenase; LHP p lipid hydroperoxides;
SOD p superoxide dismutase; totGSH p total glutathione content (including
both reduced and oxidized forms).
a
Lognormal distribution.
b
Normal distribution.
c
CAT activity of summer birds was below the detection limit.
GSH/GSSG ratio (table 4; fig. 4A, 4B). In wintering barn swallows, the completion of molt was positively associated with
GST and LHP (table 4; fig. 5A, 5B).
Discussion
The parameters describing biotransformation activity and redox state differed significantly among seasons. Values were especially high in winter barn swallows but were also elevated in
autumn barn swallows (i.e., before spring and autumn migrations). Values for birds that had recently arrived to their breeding grounds resembled values for birds captured during the
breeding period. EROD and GST activities of barn swallows
were highest in winter. A possible explanation for high GST
levels may be the energy-demanding, active molt in wintering
barn swallows. This is suggested by the fact that birds with
nearly completed molt had lower GST activities than did those
in a more active molting phase. GST can also be used in metabolizing organic hydroperoxides, such as LHP (levels were
also high in actively molting birds), which is important in
renewing of feathers (Hayes et al. 2005; Halliwell and Gutteridge
2007). An alternative explanation would be that toxic compounds increasing biotransformation activity, such as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), dioxin-like compounds or their metabolites, may
be present in the environment of wintering areas. Earlier studies
have found increased EROD activity in tree swallows and wrens
after exposure to PAHs (Bishop et al. 1999; Custer et al. 2001)
as well as in nestlings of barn swallows and black guillemots
after exposure to PCBs (Kuzyk et al. 2003; Custer et al. 2006).
Wintering barn swallows in South Africa were captured along
a river that runs through rural areas with intensive agricultural
Figure 1. Seasonal differences (estimated marginal mean Ⳳ 95% confidence interval) in enzyme activity of ethoxyresorufin-O-deethylase
(EROD; A); glutathione S-transferase (GST; B); total glutathione content, including reduced and oxidized forms (total GSH; C); ratio between
reduced and oxidized form of glutathione (GSH/GSSG ratio; D); and lipid hydroperoxide (LHP; E; logarithmic Y-axis). Mean values with the
same lowercase letter are not significantly different (Tukey’s test).
152
Figure 2. Seasonal differences (estimated marginal mean Ⳳ 95% confidence interval) in enzyme activity of superoxide dismutase (SOD; A),
catalase (CAT; B), glutathione peroxidase (GP; C), glutathione reductase (GR; D), and glucose-6-phosphate dehydrogenase (G6PDH; E). Means
with the same lowercase letter are not significantly different (Tukey’s test).
153
154 Raja-aho, Kanerva, Eeva, Lehikoinen, Suorsa, Gao, Vosloo, and Nikinmaa
Table 3: Component matrix of the principal component
(PC) analysis used to discriminate patterns of variation in
the measured parameters
Variable
Eigenvalues:
Total
Percent of variance
Cumulative %
Factor loadings:
totGSH
GST
Residual mass
EROD
LHP
SOD
G6PDH
GR
GSH/GSSG ratio
GP
Fat
PC1
PC2
PC3
2.795
25.405
25.405
2.314
21.036
46.442
1.120
10.186
56.628
.732
.707
⫺.635
.631
.508
.412
.349
.348
⫺.463
.065
⫺.260
.252
⫺.371
.464
.317
⫺.059
.339
.702
.687
.599
⫺.376
.462
.249
.150
.237
.041
.416
⫺.295
.098
⫺.380
.006
.336
.672
preparation for migration. However, its low activity, together
with the elevated GR activity, explains the high GSH/GSSG ratio
in autumn birds. One possibility is that the increased GSH/
GSSG ratio is related to the hyperphagy of premigratory birds.
Before the migration flight, birds are fuelling mainly with fats
(McWilliams et al. 2004), and high caloric intake is known to
cause oxidative conditions (Halliwell and Gutteridge 2007),
which can be effectively handled if a high GSH/GSSG ratio is
maintained. Another possibility is that the increased GSH/
GSSG ratio is in anticipation of the need to tolerate oxidative
stress in the wintering grounds. If this is the case, the finding
suggests that the oxidative conditions in the wintering grounds
are not caused by human-induced pollution, because one would
not expect genetic adaptation to occur in so few generations.
In studies involving great tits (Norte et al. 2009), the GP activities were higher during winter than during other seasons,
which would fit our observations. In barn swallows, GP levels
were also high in spring and summer birds, compared with
levels in autumn birds. The pattern of GP activity may, however,
be species specific and differ between migratory and sedentary
birds. In winter, great tits have to deal with cold weather and
Note. Loadings highlighted in bold represent the highest absolute value of
the parameter when considering different PCs. EROD p ethoxyresorufin-Odeethylase; GP, glutathione peroxidase; GR p glutathione reductase; GSH p
reduced glutathione; GSSG p oxidized glutathione; GST p glutathione Stransferase; G6PDH p glucose-6-phosphate dehydrogenase; LHP p lipid
hydroperoxide; SOD p superoxide dismutase; totGSH p total glutathione
content (including both reduced and oxidized forms).
activities as well as sources of municipal waste. In addition,
upriver is an area with several gold mines. It is also reported
that the PCB content of the air is higher in South Africa than
in Europe (Pozo et al. 2006). It is therefore possible that the
birds captured in South Africa were exposed to higher levels
of toxicants than were the birds captured in Finland. In future
studies, it would be important to measure the levels of organic
pollutants.
The intensive biotransformation metabolism of wintering
birds is one likely reason for them to have experienced oxidative
stress. The low GSH/GSSG ratio and high GSH and LHP levels
of wintering barn swallows support this notion. Increased GR
and G6PDH activities of winter and autumn birds appear to
be mainly associated with preparation for migration or with
increasing fat reserves in general. The latter enzyme is involved
in nicotinamide adenine dinucleotide/reduced form of nicotinamide adenine dinucleotide cycling in the pentose phosphate
pathway, providing reducing power for GR. Increased activities
or changed levels of all these biomarkers are associated with
oxidative stress (Halliwell and Gutteridge 2007). With regard
to totGSH, an increase (Alonso-Alvarez et al. 2008), no change
(Rattner et al. 2000; Hilscherova et al. 2003), and a decrease
(Costantini and Bonadonna 2010) in oxidative stresses have
been reported, indicating that it is not a suitable parameter for
demonstrating the occurrence of oxidative conditions.
On the basis of the present results, it is difficult to suggest
a role for GP activity changes in life-history traits, such as
Figure 3. Two first principal components (PCs) of measured liver biotransformation and oxidative stress markers (total glutathione content, including reduced and oxidized forms; glutathione S-transferase,
ethoxyresorufin-O-deethylase, lipid hydroperoxide, superoxide dismutase, glucose-6-phosphate dehydrogenase, glutathione reductase, ratio between reduced and oxidized form of glutathione, and glutathione
peroxidase) and condition measures (residual mass and fat score) in
barn swallows, by season. Wintering barn swallows had high oxidative
damage and high biotransformation enzyme activities, spring and summer birds had low biotransformation activity and intermediate oxidative damage, and autumn birds had low biotransformation activity
and oxidative stress but high redox enzyme activities. The PC loadings
are shown in table 3.
Seasonal Variation in Redox State and Biotransformation Activity in Birds
155
Table 4: Generalized linear models for the relationship between the biotransformation activity, redox
state, and body condition in the barn swallow during the annual cycle
Dependent variable, independent variable(s)
Season
Slope Ⳳ SE
F (df)
P
a
GST:
Completion of molt
LHP:b
Residual mass (g)
Completion of molt
totGSH:b
Size of testis (mm)
GSH/GSSG ratio:b
Size of testis (mm)
GR:a
Residual mass (g)
Fat score
G6PDH:b
Residual mass (g)
Fat score
Winter
.0004 Ⳳ .0001
6.79 (1, 23)
.0158
Spring
Winter
⫺.06 Ⳳ .018
.02 Ⳳ .004
9.60 (1, 21)
14.86 (1, 23)
.0054
.0008
Summer
.3 Ⳳ .09
9.24 (1, 20)
.0065
Summer
.6 Ⳳ .19
9.24 (1, 20)
.0065
4.72 (1, 21)
6.64 (1, 20)
.0413
.0180
31.94 (1, 23)
11.67 (1, 20)
!.0001
Spring
Summer
Winter
Summer
⫺.0004 Ⳳ .0002
.001 Ⳳ .0004
.1 Ⳳ .024
.3 Ⳳ .079
.0027
Note. GR p glutathione reductase; GSH p reduced glutathione; GSSG p oxidized glutathione; GST p glutathione Stransferase; G6PDH p glucose-6-phosphate dehydrogenase; LHP p lipid hydroperoxides; num df p numerator degrees of
freedom; totGSH p total glutathione content (including both reduced and oxidized forms).
a
Normal distribution.
b
Lognormal distribution.
short days. Norte et al. (2008a) hypothesized that the metabolism of great tits is slower during cold weather. Regardless of
the reason, increased biotransformation activity can cause ROS
levels to increase (Schlezinger et al. 2006), as was the case with
wintering barn swallows. High SOD and CAT activities of winter birds may also be related to the biotransformation of toxic
compounds. Both enzymes are particularly important in ROS
regulation, and the low activities of SOD in spring and summer
birds could suggest differences in redox signaling between
breeding and migratory birds.
Reproduction does not seem to change the redox state in
summer birds, compared with that in birds that have not yet
started reproducing (spring birds), although one should recall
that we could not determine the exact breeding status of summer birds. One possible reason for this is that reproduction of
barn swallows is not energetically so costly (relative to available
resources) that it would generate any imbalances. Another possibility is that some (spring) birds may have recovered from
migration and prepared for reproduction in May, when they
were captured, because they had been in Finland for 2–3 wk
already. Notably, most studies of exercise and oxidative stress
have been performed with captive birds unaccustomed to longdistance flights (Costantini et al. 2008; Larcombe et al. 2010),
which makes comparisons with our findings difficult. Marked
sex differences have been observed with reproduction-induced
oxidative conditions (Alonso-Alvarez et al. 2004; Wiersma et
al. 2004; Bertrand et al. 2006). This may be associated with
marked differences in energy gain and allocation between sexes.
Because our spring and summer groups consisted mostly of
males, we cannot compare the sex effect during the breeding
period. Testosterone elevated oxidative stress in zebra finch
(Alonso-Alvarez et al. 2007) and red-legged partridge (AlonsoAlvarez et al. 2008). In our study, the males with larger testis
had higher totGSH and GSH/GSSG ratio than did males with
smaller testis, indicating a greater reduction in redox status.
Testis size was negatively related to the capture date; for birds
captured late in the summer, the size of the testis was smaller.
The size of the testis is not necessarily related to testosterone
production (Moore et al. 2002), but larger testis size suggests
that sperm production is effective (Møller 1988). This could
indicate that males with more reducing power to cope against
ROS might have better reproduction success than their conspecifics with lower GSH/GSSG ratio.
In a study by Franson et al. (2002), molt increased the activity
of GR and GP, compared with the activity in nonmolting birds.
In our study, we found no elevation in GR or GP activity in
molting birds during winter. However, overall, both GR and
GP activities in wintering birds were high. GR activity of winter
birds, again, might be more related to preparation for migration
than to molt, because the autumn birds had values that were
similar to those for the winter birds. GST and LHP, which were
both associated with the completion of molt, are metabolically
connected. GST can metabolize organic hydroperoxides by using reduced glutathione (Hayes et al. 2005; Halliwell and Gutteridge 2007). One hypothesis for the relationship between
molt, GST, and LHP is that, during feather growth, LHP is
formed and GST metabolizes it. However, Jenni-Eiermann and
Jenni (1996) did not find an association between lipid metabolites and molting in a comparison of postbreeding, migrating,
and molting birds.
156 Raja-aho, Kanerva, Eeva, Lehikoinen, Suorsa, Gao, Vosloo, and Nikinmaa
Figure 4. Relationship between the size of testis and the redox state parameters (A) total glutathione content, including reduced and oxidized
forms, and (B) ratio between reduced and oxidized form of glutathione (GSH/GSSG ratio) in the barn swallow in summer.
Redox parameters of birds differed between winter, spring
and summer, and autumn groups. Biotransformation activity
and indications of oxidative stress were high in winter birds,
whereas most of the redox enzymes were on an intermediate
level. The spring and summer birds were profiled with low
levels of redox enzyme and biotransformation activity and exhibited intermediate oxidative stress. In the autumn, barn swallows had very little biotransformation activity, and they seemed
to have low oxidative stress even though some of the redox
enzymes (SOD, GR, and G6PDH) were highly active. It can be
assumed that low enzyme activity and intermediate oxidative
stress are associated with preparation for breeding or breeding
itself, whereas high activity of some enzymes is associated with
energetic preparation for migration.
In environmental monitoring, it is important to be aware of
the annual cycles and to take them into account in sampling
for physiological parameters. Also, studying the reasons behind
these changes helps us to understand the mechanisms of redox
regulation. To be able to generalize the results of the seasonal
changes in redox parameters across birds, more bird species
should be studied, because factors such as migratory behavior
(resident-migratory) or the developmental stage of the organism may affect the response. The best way to detect and study
oxidative stress is to use several different biomarkers, because
the interaction between various antioxidants is very important
in the overall level of defense (Monaghan et al. 2009). In the
case of the barn swallow, the knowledge of exposure to contaminants in the wintering grounds in South Africa would help
Figure 5. Relationship between the completion of molt (days left for molt completion) and (A) biotransformation activity of glutathione Stransferase (GST) and (B) redox state parameter lipid hydroperoxide (LHP) in wintering barn swallows.
Seasonal Variation in Redox State and Biotransformation Activity in Birds
in interpreting which changes are normal seasonal fluctuations
and which are caused by xenobiotic biotransformation (Pozo
et al. 2006).
Acknowledgments
We are grateful for valuable comments by two anonymous
reviewers. Kirsi Reponen, Raimo Hyvönen, Maj Björk, Ari Karhilahti, Jorma Nurmi, Jouni Saario, the Nylund family, Andre
Vosloo, Riaan Booysen, and Rina Booysen are thanked for their
help for assistance during capture and handling procedures.
This study was financed by the Emil Aaltonen Foundation
(S.R.), the Kone Foundation (M.K.), Academy of Finland (project 8119367 to T.E. and Centre of Excellence to M.N.), and
the University of Turku (Centre of Excellence to M.N.).
Literature Cited
Alonso-Alvarez C., S. Bertrand, G. Devevey, J. Prost, B. Faivre,
and G. Sorci. 2004. Increased susceptibility to oxidative stress
as a proximate cost of reproduction. Ecol Lett 7:363–368.
Alonso-Alvarez C., S. Bertrand, B. Faivre, O. Chastel, and G.
Sorci. 2007. Testosterone and oxidative stress: the oxidation
handicap hypothesis. Proc R Soc B 274:819–825.
Alonso-Alvarez C., L. Perez-Rodriguez, R. Mateo, O. Chastel,
and J. Vinuela. 2008. The oxidation handicap hypothesis and
the carotenoid allocation trade-off. J Evol Biol 21:1789–1797.
Ambrosini R., A.P. Møller, and N. Saino. 2009. A quantitative
measure of migratory connectivity. J Theor Biol 257:203–
211.
Beckman K.B. and B.N. Ames. 1998. The free radical theory
of aging matures. Physiol Rev 78:547–581.
Bertrand S., C. Alonso-Alvarez, G. Devevey, B. Faivre, J. Prost,
and G. Sorci. 2006. Carotenoids modulate the trade-off between egg production and resistance to oxidative stress in
zebra finches. Oecologia 147:576–584.
Bishop C.A., N.A. Mahony, S. Trudeau, and K.E. Pettit. 1999.
Reproductive success and biochemical effects in tree swallows
(Tachycineta bicolor) exposed to chlorinated hydrocarbon
contaminants in wetlands of the Great Lakes and St.
Lawrence River basin, USA and Canada. Environ Toxicol
Chem 18:263–271.
Bou R., R. Codony, A. Tres, E.A. Decker, and F. Guardicila.
2008. Determination of hydroperoxides in foods and biological samples by the ferrous oxidation-xylenol orange
method: a review of the factors that influence the method’s
performance. Anal Biochem 377:1–15.
Bradford M.M. 1976. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle
of protein-dye binding. Anal Biochem 72:248–254.
Burke M.D. and R.T. Mayer. 1974. Ethoxyresorufin: direct
fluorimetric assay of a microsomal o-dealkylation which is
preferentially inducible by 3-methylcholanthrene. Drug Metab Dispos 2:583–588.
Cohen A.A., K.J. McGraw, and W.D. Robinson. 2009. Serum
157
antioxidant levels in wild birds vary in relation to diet, season,
life history strategy, and species. Oecologia 161:673–683.
Costantini D. 2008. Oxidative stress in ecology and evolution:
lessons from avian studies. Ecol Lett 11:1238–1251.
Costantini D. and F. Bonadonna. 2010. Patterns of variation
of serum oxidative stress markers in two seabird species.
Polar Res 29:30–35.
Costantini D., M. Cardinale, and C. Carere. 2007. Oxidative
damage and anti-oxidant capacity in two migratory bird species at a stop-over site. Comp Biochem Phys C 144:363–371.
Costantini D., G. Dell’Ariccia, and H.P. Lipp. 2008. Long flights
and age affect oxidative status of homing pigeons (Columba
livia). J Exp Biol 211:377–381.
Custer T.W., C.M. Custer, K. Dickerson, K. Allen, M.J. Melancon, and L.J. Schmidt. 2001. Polycyclic aromatic hydrocarbons, aliphatic hydrocarbons, trace elements, and monooxygenase activity in birds nesting on the North Platte River,
Casper, Wyoming, USA. Environ Toxicol Chem 20:624–631.
Custer T.W., C.M. Custer, B.L. Goatcher, M.J. Melancon, C.W.
Matson, and J.W. Bickham. 2006. Contaminant exposure of
barn swallows nesting on Bayou D’Inde, Calcasieu Estuary,
Louisiana, USA. Environ Monit Assess 121:543–560.
Finkel T. and N.J. Holbrook. 2000. Oxidants, oxidative stress
and the biology of ageing. Nature 408:239–247.
Franson J.C., D.J. Hoffman, and J.A. Schmutz. 2002. Blood
selenium concentrations and enzyme activities related to glutathione metabolism in wild emperor geese. Environ Toxicol
Chem 21:2179–2184.
Ginn H.B. and D.S. Melville. 1983. Moult in birds. BTO Guide
19. BTO, Hertfordshire.
Halliwell B. and J.M.C. Gutteridge. 2007. Free radicals in biology and medicine. Oxford University Press, Oxford.
Hayes J.D., J.U. Flanagan, and I.R. Jowsey. 2005. Glutathione
transferases. Annu Rev Pharmacol 45:51–88.
Hilscherova K., A. Blankenship, K. Kannan, M. Nie, L.L. Williams, K. Coady, B.L. Upham, J.E. Trosko, S. Bursian, and
J.P. Giesy. 2003. Oxidative stress in laboratory-incubated
double-crested cormorant eggs collected from the Great
Lakes. Arch Environ Con Tox 45:533–546.
Jenni L. and R. Winkler. 1994. Moult and ageing of European
passerines. Academic Press, London.
Jenni-Eiermann S. and L. Jenni. 1996. Metabolic differences
between the postbreeding, moulting and migratory periods
in feeding and fasting passerine birds. Funct Ecol 10:62–72.
Kaiser A. 1993. A new multi-category classification of subcutaneous fat deposits of songbirds. J Field Ornithol 64:246–
255.
Koivula M.J. and T. Eeva. 2010. Metal-related oxidative stress
in birds. Environ Pollut 158:2359–2370.
Kuzyk Z.Z.A., N.M. Burgess, J.P. Stow, and G.A. Fox. 2003.
Biological effects of marine PCB contamination on black
guillemot nestlings at Saglek, Labrador: liver biomarkers.
Ecotoxicology 12:183–197.
Larcombe S.D., J.S. Coffey, D. Bann, L. Alexander, and K.E.
Arnold. 2010. Impacts of dietary antioxidants and flight
158 Raja-aho, Kanerva, Eeva, Lehikoinen, Suorsa, Gao, Vosloo, and Nikinmaa
training on post-exercise oxidative damage in adult parrots.
Comp Biochem Physiol B 155:49–53.
Larcombe S.D., C.A. Tregaskes, J.S. Coffey, A.E. Stevenson, L.
Alexander, and K.E. Arnold. 2008. The effects of short-term
antioxidant supplementation on oxidative stress and flight
performance in adult budgerigars Melopsittacus undulatus. J
Exp Biol 211:2859–2864.
Long J.A. and R.L. Holberton. 2004. Corticosterone secretion,
energetic condition, and a test of the migration modulation
hypothesis in the hermit thrush (Catharus guttatus), a shortdistance migrant. Auk 121:1094–1102.
Lorenzen A., J.L. Shutt, and S.W. Kennedy. 1997. Sensitivity of
common tern (Sterna hirundo) embryo hepatocyte cultures
to CYP1A induction and porphyrin accumulation by halogenated aromatic hydrocarbons and common tern egg extracts. Arch Environ Con Tox 32:126–134.
McWilliams S.R., C. Guglielmo, B. Pierce, and M. Klaassen.
2004. Flying, fasting, and feeding in birds during migration:
a nutritional and physiological ecology perspective. J Avian
Biol 35:377–393.
Møller A.P. 1988. Testes size, ejaculate quality and sperm competition in birds. Biol J Linn Soc 33:273–283.
Monaghan P., N.B. Metcalfe, and R. Torres. 2009. Oxidative
stress as a mediator of life history trade-offs: mechanisms,
measurements and interpretation. Ecol Lett 12:75–92.
Moore I.T., N. Perfito, H. Wada, T.S. Sperry, and J.C. Wingfield.
2002. Latitudinal variation in plasma testosterone levels in
birds of the genus Zonotrichia. Gen Comp Endocrinol 129:
13–19.
Nishiyama A., H. Masutani, H. Nakamura, Y. Nishinaka, and
J. Yodoi. 2001. Redox regulation by thioredoxin and thioredoxin-binding proteins. Iubmb Life 52:29–33.
Noltmann E., C.J. Gubler, and S.A. Kuby. 1961. Glucose 6phosphate dehydrogenase (zwischenferment). 1. Isolation of
crystalline enzyme from yeast. J Biol Chem 236:1225–1230.
Norte A.C., J.A. Ramos, P.M. Araujo, J.P. Sousa, and B.C. Sheldon. 2008a. Health-state variables and enzymatic biomarkers
as survival predictors in nestling great tits (Parus major):
effects of environmental conditions. Auk 125:943–952.
Norte A.C., B. Sheldon, J.P. Sousa, and J.A. Ramos. 2008b.
Repeatability and method-dependent variation of blood parameters in wild-caught great tits Parus major. Acta Ornithol
43:65–75.
Norte A.C., J.A. Ramos, J.P. Sousa, and B.C. Sheldon. 2009.
Variation of adult great tit Parus major body condition and
blood parameters in relation to sex, age, year and season. J
Ornithol 150:651–660.
Nouroozzadeh J., J. Tajaddinisarmadi, S. McCarthy, D.J. Betteridge, and S.P. Wolff. 1995. Elevated levels of authentic
plasma hydroperoxides in niddm. Diabetes 44:1054–1058.
Pozo K., T. Harner, F. Wania, D.C.G. Muir, K.C. Jones, and
L.A. Barrie. 2006. Toward a global network for persistent
organic pollutants in air: results from the GAPS study. Environ Sci Technol 40:4867–4873.
Rattner B.A., D.J. Hoffman, M.J. Melancon, G.H. Olsen, S.R.
Schmidt, and K.C. Parsons. 2000. Organochlorine and metal
contaminant exposure and effects in hatching black-crowned
night herons (Nycticorax nycticorax) in Delaware Bay. Arch
Environ Con Tox 39:38–45.
Saino N., M. Romano, R. Ambrosini, R.P. Ferrari, and A.P.
Møller. 2004. Timing of reproduction and egg quality covary
with temperature in the insectivorous Barn Swallow, Hirundo
rustica. Funct Ecol 18:50–57.
SAS Institute. 2008. SAS/STAT 9.2 User’s guide. SAS Institute,
Cary, NC.
Schlezinger J.J., W.D.J. Struntz, J.V. Goldstone, and J.J. Stegeman. 2006. Uncoupling of cytochrome P450 1A and stimulation of reactive oxygen species production by co-planar
polychlorinated biphenyl congeners. Aquat Toxicol 77:422–
432.
Steidl R.J. and L. Thomas. 2001. Power analysis and experimental design. Pp. 14–36 in S.M. Scheiner and J. Gurevitch,
eds. Design and analysis of ecological experiments. Oxford
University Press, New York.
Stohs S.J. and D. Bagchi. 1995. Oxidative mechanisms in the
toxicity of metal-ions. Free Radical Bio Med 18:321–336.
Svensson L. 1992. Identification guide to european passerines.
Fingraf, Södertälje, Sweden.
Szep T., A.P. Møller, S. Piper, R. Nuttall, Z.D. Szabo, and P.L.
Pap. 2006. Searching for potential wintering and migration
areas of a Danish Barn Swallow population in South Africa
by correlating NDVI with survival estimates. J Ornithol 147:
245–253.
———. 2007. Migratory connectivity in barn swallows and
other hirundines. J Ornithol 148:257–260.
Tanhuanpää S., T. Eeva, E. Lehikoinen, and M. Nikinmaa. 1999.
Developmental changes in 7-ethoxyresorufin-O-deethylase
(EROD) and delta-aminolevulinic acid dehydratase (ALAD) activities in three passerines. Comp Biochem Physiol C
124:197–202.
Valavanidis A., T. Vlahogianni, M. Dassenakis, and M. Scoullos.
2006. Molecular biomarkers of oxidative stress in aquatic
organisms in relation to toxic environmental pollutants. Ecotox Environ Safe 64:178–189.
Whyte J.J., R.E. Jung, C.J. Schmitt, and D.E. Tillitt. 2000. Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Crit Rev Toxicol 30:347–570.
Wiersma P., C. Selman, J.R. Speakman, and S. Verhulst. 2004.
Birds sacrifice oxidative protection for reproduction. Proc R
Soc B 271:S360–S363.