Environ. Sci. Technol. 2008, 42, 2668–2673
Oxidative Stress during Baltic Salmon Feeding Migration May Be Associated with Yolk-sac Fry Mortality K R I S T I I N A A . V U O R I , * ,† MIRELLA KANERVA,† ERKKI IKONEN,‡ AND MIKKO NIKINMAA† Centre of Excellence in Evolutionary Genetics and Physiology, Department of Biology, University of Turku, FI-20014 Turku, Finland, and Finnish Game and Fisheries Research Institute, Viikinkaari 4, P.O. Box 2, FI-00791 Helsinki, Finland
Received October 18, 2007. Revised manuscript received January 4, 2008. Accepted January 12, 2008.
The wild populations of salmon in the Baltic Sea suffer from yolksac fry mortality (M74). M74 mainly occurs in populations spawning in rivers flowing to the Gulfs of Bothnia and Finland. On the basis of studies with fry, M74 may be caused by oxidative stresses. Because the eggs of M74-offspring-producing females have lower thiamine and astaxanthin levels and more oxidized fatty acids than eggs of females producing healthy offspring, oxidative stresses that adult salmon experience during their feeding migration may be decisive for the development of M74. In this study we have measured several oxidative stress parameters and have evaluated both temporal and regional differences in these parameters in salmon individuals during their feeding migration. At present, salmon feeding in the Gulf of Finland and in the Bothnian Sea are affected by oxidative stress as compared to populations feeding in the Baltic Proper. Moreover, the feeding population of salmon in the central Baltic Proper suffered much more from oxidative stress in 1999 than in 2006–2007. In 1999 the incidence of M74 was higher than that expected in 2007/2008. Oxidative stresses experienced by feeding salmon may thus be behind the development of M74.
Introduction One of the most conspicuous examples of early life stage mortalities in salmonids from natural ecosystems is the yolksac fry mortality (M74) of Baltic salmon (Salmo salar). The syndrome has been designated as M74 according to the year of discovery and Swedish word miljörelaterad (environmentally induced). M74 occurs in wild and stocked salmon populations spawning in rivers flowing to the Gulfs of Bothnia (including Bothnian Sea) and Finland. Only fishes from the river Mörrumsån are affected among populations in the Baltic Proper (1–3). The incidence of the M74 syndrome is variable. In the 1990′s, 25–80% of Baltic salmon females, which ascended rivers to spawn, produced yolk-sac fry suffering from the syndrome (1). Of the fry produced, 50–80% died. In the years 2003–2005, only a few % of the salmon fry suffered * Corresponding author e-mail:
[email protected]; phone: +35823336263; fax: +35823336598. † University of Turku. ‡ Finnish Game and Fisheries Research Institute. 2668
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from the syndrome, but in 2006 a higher percentage of the fry appear to suffer from the syndrome than in previous years (1). M74 has strongly affected the self-sustaining salmon populations, as decreased parr densities were detected in the nursery areas during the periods of high occurrence (4, 5). Oxidative stresses may cause the symptoms that are part of the M74 syndrome. The effects of oxidative stress can be transmitted from the parent to the egg (6). M74 is associated with a low thiamine content in the brood fish and eggs (2, 7, 8) and with reduced levels of antioxidants, especially astaxanthin (2, 9, 10). In addition, the cellular glutathione ratio (GSH/GSSG) of the M74-fry is altered in favor of the oxidized form (11), and the activities of redox enzymes in the liver—glutathione peroxidase, glutathione reductase, and glutathione-S-transferase—are increased (11, 12). Thiamine deficiency itself has been shown to be caused by oxidative stresses (e.g., refs 13 and 14). M74 is transferred maternally. Because the eggs of M74offspring-producing females have lower thiamine and astaxanthin levels than the eggs of females producing healthy offspring (8, 10, 15, 16), and because the M74-eggs have more oxidized fatty acids than healthy ones (15, 16), oxidative stresses that adult salmon experience during their feeding migration in the Baltic Sea may be decisive for the development of M74. Karlsson et al. (17), Hansson et al. (18), Pickova et al. (16) and Ikonen (19) have suggested that changes in the properties of the food web may be key factors producing M74 by affecting the chemical composition of salmon prey. It is not known what would cause the changes in the chemical composition of salmon prey and how the changes in the chemical composition would result in, for example, decreased amounts of antioxidants in the eggs of salmon females. If oxidative stresses play a role in the development of M74, then differences in oxidative stress metabolism should be evident in actively feeding adult salmon females prior to spawning. Oxidative stresses in M74-offspring-producing female salmon may be caused either by a decreased supply of antioxidants from food or by increased biotransformation of anthropogenic or natural toxic compounds (causing oxidation) or any combination of these. We hypothetize that, during the feeding migration of salmon, decreased antioxidant supply may lead to compensatory increases in the activities of reactive oxygen species (ROS) handling enzymes and may decrease the levels of antioxidants and vitamins deposited to the eggs. Enhanced biotransformation processes may cause an increase of ROS by increasing the activity of biotransformation enzymes, decreasing the antioxidant pool, and increasing the activities of ROS-handling enzymes with the consequence that decreased levels of antioxidants and vitamins are deposited to the eggs. These changes could be caused by temporal and regional variation in the chemical composition of salmon prey. This study shows that oxidative stress parameters of feeding Baltic salmon populations differ both regionally and temporally and that the temporal differences agree with the incidence of M74.
Materials and Methods Sampling. The feeding salmon were caught from International Council for the Exploration of the Sea (ICES) subdivisions 25 (Bornholm deep), 28 (Gotland deep), 30 (Bothnian Sea), and 32 (Gulf of Finland) during the late autumn 2006/ winter 2007 (November-January) with the help of Finnish fishermen. The map of the Baltic Sea with ICES subdivisions is presented in Figure S1 of the Supporting Information. 10.1021/es702632c CCC: $40.75
2008 American Chemical Society
Published on Web 02/27/2008
TABLE 1. Properties of Sample Groups: Age, Weight, Length, and Condition Factor (Cf; weightg/lengthcm3*100) Are Shown as Means (Standard Deviation)a group 25, 28, 30, 32, 99, a
Southern Baltic Central Baltic Bothnian Sea Gulf of Finland Central Baltic
N
wild/stocked
sex F/M
age (sd), sea years
weight (sd), kg
length (sd), cm
Cf (sd)
49 9 25 30 23
22/27 8/1 7/18 1/29 N/A
36/7 8/1 20/4 18/10 N/A
1.7 (0.9) 3.0 (0.8) 1.4 (1.2) 1.0 (0.2) N/A
5.2 (2.2) 6.5 (3.1) 4.6 (2.5) 4.3 (1.1) 5.3 (3.4)
81.5 (9.5) 83.7 (10.4) 72.6 (15.3) 71.8 (5.9) 77.9 (11.7)
2.7 (1.0) 3.3 (1.4) 2.5 (1.2) 2.4 (0.5) 2.8 1.5)
N/A, information not available.
Temporal differences could be investigated with the help of samples from the Environmental Specimen Bank, Swedish Museum of Natural History, collected in November 1999 from Gotland deep (sample group 99). Hereafter, the different groups of fish are designated with the numerical value of the catching subdivision, except that the group caught in Gotland deep in November 1999 is designated 99. The fish were killed and weighed, and the livers were excised, frozen immediately in liquid nitrogen, and stored at -84 °C until measurements. The properties of the sample groups are shown in Table 1. The age (sea years) and the origin were determined from scale samples (20, 21). Cut fatfin confirmed the stocked origin in 29% of the cases. Sample Homogenates. Frozen liver pieces were crushed in liquid nitrogen with mortar. Thereafter, a piece of liver (about 1 × 0.5 cm) was homogenized in 4 mL of 0.1 M K2HPO4 + 0.15 M KCl-buffer (pH 7.4) using an Ultra Turrax homogenizer (IKA Labortechnik, Staufen, Germany) 5 times for 10 s on ice, 16 000 rpm/min. A 100 µl portion of this homogenate was pipetted to an Eppendorf tube with 10 µl of M2VP (1-methyl-2-vinylpyridiniumtrifluoromethanesulfonate) scavenger (GSH/GSSG kit, OxisResearch, Foster City, California) for the determination of oxidized glutathione (GSSG). A 50 µl portion of plain homogenate was pipetted into an Eppendorf tube for reduced glutathione (GSH) determination. Both tubes were frozen immediately in liquid nitrogen and stored at -84 °C. The rest of the homogenate was centrifuged for 15 min at 10 000g and +4 °C. The supernatant was divided into aliquots, frozen in liquid nitrogen, and stored at -84 °C until further measurements. Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay (EMSA). Frozen liver pieces were crushed in liquid nitrogen with a mortar. A piece of liver with a diameter of approximately 0.5 cm was used to prepare nuclear extract. The DNA binding of aryl hydrocarbon receptor (AhR) to dioxin response element (DRE) was evaluated from nuclear extracts using EMSA. The preparation of both nuclear extracts and EMSA were done as described by Vuori et al. (22). The method measures transcription factor complex binding to the response element in DNA but does not show the presence of specific AhR isoforms in the complex. Determinations of Enzyme Activities. The enzyme activities were measured in triplicate using 96-well microplates, which in most cases required reducing reagent volumes as compared to method instructions. The glutathione reductase (GR), glutathione peroxidise (GP), glutathione S-transferase (GST), and catalase (CAT) activities were measured with Sigma kits (Sigma Chemicals, St. Louis, Missouri, USA). The GP activity was measured using 2 mM H2O2 as a substrate. The inhibition rate of superoxide dismutase (SOD) was measured using a Fluka (Fluka, Buchs, Germany) kit and GSH/GSSG ratio using an OxisResearch (Foster City, California) kit. Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured according to Noltmann et al. (23), and ethoxyresorufin-O-deethylase (EROD) activity was measured according to Burke and Mayer (24). The protein content was determined with the Bradford method using BioRad protein assay (BioRad, Espoo, Finland)
with bovine serum albumin (Sigma) as the standard. The GP measurement was done with a Multiscan Ascent (Thermo Labsystems, Vantaa, Finland), and all other measurements with were with Victor I and II (Perkin-Elmer Wallac, Turku, Finland) microplate readers. Statistics. SPSS 12.0.1 software was used for all statistical analyses. In general, the data were not normally distributed (Shapiro-Wilk’s test for normality). Therefore, initially both parametric and nonparametric (Kruskal–Wallis) ANOVAs were used. Tamhane’s T2 test and Mann–Whitney’s U-test were used as posthoc tests. Statistical significances from Tamhane’s T2 test are reported in cases where both tests gave equal results. Principal component analysis (PCA) was conducted using nontransformed data and unrotated factor solution. Parameters included in the PCA were weight, length, SOD, CAT, GST, GR, G6PDH, GSH/GSSG ratio, GSH, GP, DRE, and EROD. Condition factor was excluded because it is calculated from weight and length. Age was excluded because it was not available for group 99. Factor scores were derived with the regression method.
Results Table 1 gives the basic properties of the sampled salmon. The proportions of fish with wild origin was 44% for fish caught in Bornholm deep (SD 25), 90% for fish caught in Gotland deep (SD 28), and 25% for fishes caught in the Bothnian sea (SD 30), but only 3% (1 fish out of 30) for fish caught in the Gulf of Finland (SD 32). Neither the origin (wild/stocked, Tables S1 and S2 of the Supporting Information) nor the size (Tables S3–6 of the Supporting Information) of fish correlated with the measured parameters. To get a complete picture of the oxidative stress status of the feeding populations of Baltic salmon, we analyzed the activities of SOD, CAT, G6PDH, GP, and GR and the ratio between reduced and oxidized glutathione (GSH/GSSG) (Figure S2a, Supporting Information). The activity of GST was analyzed as an additional indicator of changes in glutathione metabolism because it is an enzyme of phase II biotransformation process, thus functioning in the treatment of both natural and xenobiotic ligands, for example, in lipid peroxide detoxification (25, 26). The activity of EROD was analyzed to get an indication of the AhR-dependent biotransformation activity (27). As a further measure of the induction of AhR-dependent gene expression pathway, the DNAbinding of dioxin (xenobiotic) response element (DRE; Figure S2b, Supporting Information) (27) was analyzed. Individual parameters measured are shown in Figure 1. For clarity, only statistical significances between means of group 25 and the other groups are shown in the figure. The SOD activity increased in groups 30, 32, and 99 compared to group 25 (Tamhane’s T2 test, p < 0.001).The activity of CAT decreased (Tamhane’s T2 test, p < 0.001) in groups 32 and 99 compared to group 25. Pronounced oxidative stress is seen in a 4-5-fold decrease of the GSH/GSSG ratio in groups 32 and 99 compared to group 25 (Tamhane’s T2 test, p < 0.001), as a result of increased amounts of oxidized form of glutathione. The total GSH µM/mg protein decreased in VOL. 42, NO. 7, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Individual parameters measured. X-axis: sample groups. Y-axes: means ( standard error of mean. The parameter name is shown on the top of each figure. *** p < 0.001, ** p < 0.01. group 99 compared to group 25 (Tamhane’s T2 test, p < 0.001), suggesting a very chronic oxidative stress in this group. The other measured parameters also fit fish experiencing oxidative stress in group 30, but especially in groups 32 and 99. GST activity is induced 4-5-fold in groups 30, 32, and 99 compared to group 25 (Tamhane’s T2 test, p < 0.001). G6PDH activity is increased in groups 30, 32, and 99 (Mann–Whitney’s U-test, p < 0.01). GP activity increased in groups 30 and 32 compared to group 25 (Tamhane’s T2 test, p < 0.001), and GR activity increased in groups 32 and 99 (Mann–Whitney’s U-test, p < 0.05). In accordance with the observed individual variability in the fish producing offspring with M74, the GST, G6PDH, GP, and GR activities show higher within-group variation in groups 30, 32, and 99 than in group 25. The parameters measured to indicate xenobiotically induced gene expression and consecutive biotransformation did not show a uniform pattern. The GST activity was higher in groups 30, 32, and 99 than in group 25. The EROD activity was, however, significantly increased only in group 32 (Tamhane’s T2 test, p < 0.001). In contrast, EROD was significantly decreased in group 99 as compared to group 25 (Tamhane’s T2 test, p < 0.001). DRE binding was significantly increased in both groups 32 and 99 (Tamhane’s T2 test, p < 0.001). SOD, CAT, GST, GR, GSH/GSSG ratio, GSH, and DRE were also significantly different between groups 28 and 99 (Table S7, Supporting Information), representing temporal differences in the same location, Central Baltic Proper. 2670
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On the basis of principal component analysis (PCA) (Figure 2 and Figure S3, Supporting Information), the two main components explained 29% (component 1, C1) and 17% (component 2, C2) of the observed variation. Because all oxidative stress parameters measured (SOD, CAT, GST, GR, G6PDH, GSH/GSSG ratio, and GP) were included in the component explaining the biggest proportion of the variation (C1) (Table 2), it appears that regionally (and temporally) different Baltic salmon populations are characterized by different degrees of oxidative stress. Notably, EROD activity was not included in C1. As can be expected, the weight and the length of fish explain some of the total variation (forming C2; Table 2).
Discussion Oxidative stress in an organism results when the external input and metabolic production of various reactive oxygen species (ROS) exceed the organism’s capacity to handle them. Its development is discussed in detail by Halliwell and Gutteridge (26). The oxidizing compounds are handled by small-molecule antioxidants, including vitamins A, C, and E, carotenoids, reduced glutathione (GSH), and thioredoxin, and by enzymes metabolizing the compounds. Enzymes that directly handle ROS are SOD, which converts superoxide to hydrogen peroxide, GP, and CAT, which converts hydrogen peroxide to molecular oxygen and water. This enzyme battery is supplemented by enzymes, such as GR and G6PDH, providing reducing equivalents for the handling of ROS.
FIGURE 2. The different sample groups are divided in PCA. X-axis: Component 1 (C1) values (29% of variation). Y-axis: Component 2 (C2) values (17% of variation). C1 includes all oxidative stress parameters measured (SOD, CAT, GST, GR, G6PDH, GSH/GSSG ratio, and GP), and C2 measures weight and length. Specimens of wild origin are marked with -.
TABLE 2. PCA Component Matrixa
weight length SOD CAT GST GR G6PDH G ratio GSH GP DRE EROD
C1
C2
-0.162 -0.323 0.600 -0.549 0.856 0.597 0.648 -0.626 -0.183 0.518 0.521 -0.016
0.796 0.803 0.017 -0.420 -0.069 -0.044 -0.126 -0.238 -0.449 -0.246 0.397 -0.277
a
Two main components explain 29% (component 1, C1) and 17% (component 2, C2) of the variation. All of the oxidative stress parameters measured in this study (SOD, CAT, GST, GR, G6PDH, GSH/GSSG ratio, and GP) were included in the C1, and weight and length formed C2.
The parameters measured in this study and PCA results indicate that of the feeding populations of salmon studied, group 30, but especially groups 32 and 99, experienced oxidative stress. Oxidative stress increases the oxidation of glutathione to GSSG, whereby the reduced form, GSH, may eventually be depleted upon prolonged stress unless the enzyme systems involved in the redox cycling of glutathione are activated (28). The key enzymes in the cycling are GP and GR. However, GP and GR may not be activated simultaneously but more as a step-by-step process, depending on the degree of the oxidative stress experienced (Figure 3). It appears that when there is a normal balance between ROS generation and breakdown, as in groups 25 and 28, GP and GR activities are low, and the amount of GSSG is not increased. When some oxidative stress occurs, as in group 30, GP is activated, and GSSG may start to accumulate later on. When the oxidative stress is severe, GP and GR are both activated, and the GSH/GSSG ratio is low due to high amounts of GSSG. This is the situation of group 32 salmon. When the stress is severe and prolonged, as in group 99, the enzymes, especially GP, start to be inhibited, and the total amount of glutathione starts to decrease.
FIGURE 3. A possible scenario for glutathione metabolism in the different groups analyzed. When there is a normal balance between ROS generation, and antioxidant defense mechanisms (groups 25 and 28), enzyme activities are low, and the amount of GSSG is not increased. In intermediate conditions of oxidative stress (group 30), GP is activated, and GSSG may start to accumulate later on. Upon severe oxidative stress, GP and GR are both activated, and the GSH/GSSG ratio is low due to high amounts of GSSG (group 32). When the stress is severe and prolonged (group 99), some enzyme activities will eventually decrease, and the total amount of GSH starts to drop. The mere differences in the proportions of prey species — herring, sprat, and stickleback — eaten do not explain the variable incidence of M74 (17–19). Instead, properties of the food web, affecting the chemical composition of salmon prey, may be the key factor that causes M74 by affecting the redox status of feeding salmon populations and, consequently, the amounts of antioxidants and vitamins deposited to the eggs of female salmon. Changes in the food web could be associated with recent eutrophication and decreasing salinity of the Baltic Sea. Both changes in salinity (29) and eutrophication (30) affect phytoplankton communities and their cellular levels of antioxidants. In addition, a general shift from a more neritic toward a more limnic zooplankton community has occurred because of the decreased inflow of saline water (31, 32), and the zooplankton species composition in the Baltic Sea varies according to salinity gradients (33). The regional and annual changes in phyto- and zooplankton may be reflected in changes in the growth and the quality of prey species eaten by Baltic salmon. In SDs 30 and 32, specimens of the main salmon prey species, herring, have a lower condition factor than herring in the southern VOL. 42, NO. 7, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Baltic Proper because of the lower quality food (limnic zooplankton species) available (19). Neritic zooplankton species, which are more abundant in the southern and central Baltic Proper, may also offer a better source of astaxanthin than limnic species (34). Because carotenoids and thiamine in the salmon diet originate from phytoplankton, any change in the composition of plankton communities could decrease the production or transformation of these compounds and their consecutive transfer to salmon via plankton and prey fish (35). However, the concentrations of thiamine in the Baltic Sea plankton do not differ from other coastal areas (35), and the concentrations in sprat and herring are adequate for salmon diet (36). Oxidative stresses may deplete the general antioxidant pool of brood fishes (26, 28) and ultimately deplete their thiamine levels by activating the pentose phosphate pathway (PPP), where transketolase requires thiamine as a cofactor (14). Notably, in our data the activity of G6PDH, the rate limiting enzyme of PPP, was increased whenever oxidative stress was apparent. If GST and EROD activities and the DNA-binding of AhR to the dioxin response element (DRE) are taken to indicate increased xenobiotically induced gene expression and consecutive biotransformation, then especially fish from the Gulf of Finland are affected. The EROD activity was significantly increased only in group 32 but, in contrast, significantly decreased in group 99 as compared to group 25. The difference need not indicate that the xenobiotic gene expression pathway is not activated. The measurement of EROD activity is measuring enzyme function, and it may decrease even if both the transcription of the gene and its translation to the protein are increased. In the case of EROD, the enzyme activity is usually increased by various organochlorine pollutants (e.g., Whyte et al. (27)), but may be decreased in the natural environment after a chronic severe contamination (37). Hansson et al. (38) found no correlation between EROD induction and organochlorine toxicants in salmon. The increased amount of AhR and CYP1A transcription was, however, correlated with the toxicant load. Although EROD induction was not generally associated with the oxidative stress parameters measured in this study, the importance of toxicants in the development of M74 cannot be excluded. Previously, conclusions about the association of toxicants with M74 have been contradictory (6). The data of Isosaari et al. (39) demonstrate that salmon from the northern Baltic Sea (including the Gulf of Finland, the Bothnian Sea and the Bothnian Bay) have higher fresh weight concentrations of PCDD/Fs (polychlorinated dibenzop-dioxins and dibenzofurans), PCBs (polychlorinated biphenyls), PCNs (polychlorinated naphthalenes) and PBDEs (polybrominated diphenyl ethers) than fish from the southern Baltic Proper. This is in line with our findings about higher oxidative stress in the Bothnian Sea and the Gulf of Finland if one supposes that oxidative stress is largely the result of toxicant load. In our study, the DNA binding of AhR (DRE) was increased in the groups 32 and 99, affected most by oxidative stress. Elevated GST activities were also detected in all groups (30, 32, 99) affected by oxidative stress. In contrast, fish caught from the Bothnian Sea (group 30) did not have elevated DRE or EROD. However, the increased DNA binding of AhR in the present data cannot be regarded merely as a response to toxicants as recent information shows that the pathway is involved in the regulation of the expression of various other genes, involved in, e.g., biotransformation and oxidative stress defense. For example, AhR status (present/absent) alone affected the expression of 392 probe sets in mouse liver (40). Oxidative stresses experienced by adult, feeding salmon may correlate with the yolk-sac fry mortality of Baltic salmon. Although sampling of feeding populations does not allow the reproduction success of specimens sampled to be 2672
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evaluated directly, this conclusion is supported by temporal comparison of redox status of group 99 with groups 28 and 25. In 1999 and 2000, the proportion of M74-offspringproducing females in Finnish rivers was approximately 60 and 40% (1). In 2005 and 2006 the proportions were
