Environmental Toxicology and Chemistry, Vol. 34, No. 10, pp. 2403–2408, 2015 # 2015 SETAC Printed in the USA
MERCURY AND SELENIUM CONCENTRATIONS IN SKELETAL MUSCLE, LIVER, AND REGIONS OF THE HEART AND KIDNEY IN BEARDED SEALS FROM ALASKA, USA LUCERO CORREA,*y J. MARGARET CASTELLINI,z LORI T. QUAKENBUSH,x and TODD M. O’HARAyk yDepartment of Chemistry and Biochemistry, University of Alaska Fairbanks, Fairbanks, Alaska, USA zSchool of Fisheries and Ocean Sciences, University of Alaska Fairbanks, Fairbanks, Alaska, USA xAlaska Department of Fish and Game, Fairbanks, Alaska, USA kDepartment of Veterinary Medicine, University of Alaska Fairbanks, Fairbanks, Alaska, USA (Submitted 13 December 2014; Returned for Revision 15 February 2015; Accepted 20 May 2015) Abstract: Mean concentrations of total mercury ([THg]) and selenium ([TSe]) (mass and molar-based) were determined for 5 regions of the heart and 2 regions of the kidney of bearded seals (Erignathus barbatus) harvested in Alaska, USA, in 2010 and 2011. Mean [THg] and [TSe] of bearded seal liver and skeletal muscle tissues were used for intertissular comparison. The Se:Hg molar ratios were used to investigate elemental associations and potential antioxidant protection against Hg toxicosis. Age was an important factor in [THg] and Se:Hg molar ratios in heart and kidney. Small but statistically significant differences in mean [THg] occurred among some of the 5 heart regions (p < 0.05). Mean [THg] was highest in liver, 3.057 mg/g, and lowest in heart left ventricle, 0.017 mg/g. Mean [THg] ranked: liver > kidney cortex > kidney medulla > skeletal muscle > heart left ventricle (p < 0.001). Mean [TSe] was highest in liver, 3.848 mg/g, and lowest in heart left ventricle, 0.632 mg/g. Mean [TSe] ranked: liver > kidney cortex > kidney medulla > skeletal muscle > heart left ventricle (p < 0.001). The Se:Hg molar ratios were significantly greater than 1.0 in all tissues (p < 0.001) and represented baselines for normal [TSe] under relatively low [THg]. Mean Se:Hg molar ratios ranked: heart left ventricle > kidney medulla > kidney cortex (p < 0.001). Environ Toxicol Chem 2015;34:2403–2408. # 2015 SETAC Keywords: Mercury
Selenium
Biomonitoring
Molar ratio
Bearded seal
some forms of Hg in the liver and kidney are well known for humans as well as some other animals, whereas methylated forms of Hg are well-known neurotoxins. Liver and kidney are major sites of inorganic mercury (Hg2þ) accumulation, which in many mammals can result in hepato- or nephrotoxicity, respectively [4,12,13]. Lesions in various regions of the liver and kidney have been associated with [THg] above the toxic threshold (60 mg/g wet wt) for marine mammals [12,14,15]. Some forms of Hg can have interactive effects on heart and kidney function, resulting in hypertension or heart disease [13,16,17]. It has been hypothesized that Hg2þ has the potential to interact with sulfur-containing compounds, such as glutathione (GSH), to induce high blood pressure and increase the risk for hypertension and vascular disease [18,19]. Hypertension is thought to be an indirect impact of Hg on the cardiovascular system [18,19]. In many marine mammal studies, [THg] and total concentrations of Se ([TSe]) are positively correlated [20,21]. It has been suggested that a molar Se:Hg ratio significantly greater than 1.0 in certain tissues (such as liver and kidney) may be beneficial to the formation of insoluble HgSe compounds and detoxifying MeHgþ [21]. Selenium concentrations tend to be higher in some piscivorous marine mammals than in terrestrial mammals. In addition, Se can act directly as an antioxidant or as a component of key antioxidants [22,23]. Because of the potential antioxidant role of Se and its direct interactions with Hg, it is also important to understand the distribution of both [THg] and [TSe] in tissues such as heart and kidney. Distributions of Hg and Se in specific regions of the heart and kidney are not usually reported and can prove beneficial in determining biochemical and histopathological effects of mercury. The potential for Hg distribution to differ by region of the heart and kidney is in part the result of the variation in vascularization and concentration of sulfur-containing
INTRODUCTION
Bearded seals (Erignathus barbatus) are 1 of 3 species of sea ice–associated seals used for food in subsistence-based communities in northern Alaska, USA. Population health and status of bearded seals have been monitored since 1962 by the Alaska Department of Fish and Game in cooperation with the Alaska Native subsistence harvest. As an Arctic species, bearded seals are exposed to contaminants such as perfluorinated pollutants [1], organochlorines, and heavy metals such as Hg [2,3]. Numerous studies have been published and many are ongoing related to Hg and Se in vertebrates of the Arctic [4–7]. In particular, Dietz et al. [4] outlined that Hg concentrations in numerous species (such as polar bears, whales, and seals) across the Arctic exceed various guidelines of concern for adverse effects, and they may be increasing over time. Basu et al. [8,9] specifically addressed some of the potential neurotoxicological outcomes and mechanisms of action of some arctic mammals. Thus, it is very relevant to assess Hg and Se concentrations in ice seals, including bearded seals. These animals are important sentinels for assessing potential adverse effects (suite of potential target tissues) and for biomonitoring over space and time (tissues known to be reliably sampled with easily detected concentrations) as outlined in Dietz et al. [4]. Similar to other piscivorous marine species [5], bearded seals bioaccumulate and biomagnify monomethylmercury (MeHgþ) in tissues such as liver, kidney, and skeletal muscle. In arctic seals, especially older piscivores, liver has higher total Hg concentrations ([THg]) than kidney [10,11]. The toxic effects of * Address correspondence to [email protected] Published online 26 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.3079 2403
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compounds, such as GSH and myoglobin, known to bind to various forms of Hg [24–26]. Heart morphology is relatively similar across mammalian species; however, kidney morphology varies (for example, nonreniculated and multireniculated). Seals have multireniculated kidneys, which contain clusters of cortical and medullary structures within each kidney [27,28]. The present study measured [THg] in different regions (intratissular comparisons) of the heart (each chamber wall and the septum) and kidney (cortex and medulla), and compared these values with [THg] in liver and skeletal muscle tissue (intertissular comparisons) [2,3] to understand the regional distribution of [THg] within and among seal tissues. In addition, we measured [TSe] and Se:Hg molar ratios in heart left ventricle and kidney regions (cortex and medulla) to assess regional distribution and relative potential defense mechanisms of Se against Hg toxicosis. MATERIALS AND METHODS
Sample collection
Heart, kidney, skeletal muscle, and liver samples were collected from bearded seals (age kidney medulla > heart (p < 0.001). In all 3 tissues, [TSe] > [THg] (p < 0.001). Statistically significant differences are indicated by different letters.
studies [2,3,10,31]. Even though [THg] is highest in liver, it is known that in adult seals approximately 90% is in the form of Hg2þ, most of which (approximately 53%) is bound to some form of Se and therefore likely to be biologically inactive [2,10]. Liver [THg] and [TSe] in the present study were slightly lower but within the range ([THg] range ¼ 0.64–20.44 and [Se] range ¼ 0.75–23.20 [2]; [THg] range ¼ 1.92–5.22 and [Se] range, 3.40–6.50 [3]) of previous bearded seal studies. Skeletal muscle of bearded seals had greater [THg] than did heart (Table 1). This may be because skeletal muscle has more myoglobin [32,33], which stores and diffuses oxygen within muscle cells [34]. During diving, seals preferentially direct blood to the heart and brain, which maintains an adequate oxygenated blood supply to the heart during long periods of hypoxia, thus reducing the need for heart muscle to use oxygen stored in myoglobin [23]. Myoglobin’s sulfur-containing amino acid cysteine makes it an ideal molecule for binding MeHgþ. Thus, myoglobin may be the reason that 80% or more of THg in skeletal muscle is in the form of MeHgþ [2,35]. The [THg] in skeletal muscle does not vary with age, making this an ideal tissue for monitoring MeHgþ exposure because it eliminates, or at least limits, age as a confounding factor relative to tissues such as liver and kidney. Skeletal muscle [THg] and [Se] in the present study were within the ranges found in Dehn et al. [2].
Mean [THg] and [TSe] in bearded seal heart tissue have not been previously reported. Although the mean [THg] among the 5 regions of the bearded seal heart were different (Figure 1), this result is not likely to be of toxicological or biological significance because the concentrations were low in all regions. In addition, the molar [TSe] was greater than the molar [THg], indicating a potential protective interaction. However, when sampling the heart to determine [THg], it may be important to standardize where the sample is taken (e.g., sample only left ventricle) to reduce the effects that this small variability may have on spatial (populations) and temporal comparisons. In the kidney, we found higher [THg] in cortex than medulla (Figure 2), which was similar to what is reported for terrestrial animals [13,25]. Transport of Hg to the kidney is mediated by sulfur-containing compounds in the blood, such as GSH, albumin, and cysteine. Glutathione has a higher absorption rate into the proximal tubules of the cortex, which likely facilitates the uptake and subsequent accumulation of Hg in the kidney cortex [25]. The disparity between concentrations in the cortex and medulla in the multireniculated kidney suggests that standardized protocols for sampling marine mammal kidneys may be needed to reduce sample variability. Such a protocol might include combining an equal amount of cortex and medulla for analysis, analyzing only the cortex or medulla, or analyzing both the cortex and medulla separately. Sampling cortex and medulla separately may be a problem if a study is focused on young seals because they have smaller renicules, making it difficult to distinguish and dissect cortex and medulla. In adults, however, specific subsampling of cortex and medulla is fairly easy. Thus, the objective of a particular study will dictate the sample protocol that is most appropriate. That [THg] varies with age in the cortex but not in the medulla could be useful in some study designs. For normal biomonitoring purposes, however, it may be best to sample equal proportions of cortex and medulla in animals of a known age to compare [THg] across age groups and for improving temporal and spatial analyses of [THg] in seal monitoring efforts. Kidney [THg] and [TSe] are generally not reported for both cortex and medulla but rather a subsample of the whole kidney. In the present study, analyzing kidney [THg] and [TSe] for cortex and medulla was not different from using whole kidney for comparisons with muscle and heart [2]. Se:Hg molar ratios
Figure 3. Mean ( standard deviation) Se:Hg molar ratio in heart left ventricle, kidney cortex, and kidney medulla tissue of bearded seals. Statistically significant differences among tissues were as follows: heart > kidney medulla > kidney cortex (p < 0.001), as indicated by different letters.
Differences in relative distribution of [TSe] and [THg] (Figure 2) between kidney medulla and heart result in differences in mean Se:Hg molar ratios within these tissues. Mean Se:Hg molar ratios in heart left ventricle (skeletal muscle mean Se:Hg ratio ¼ 50.04) and kidney medulla were greater than that in kidney cortex (liver mean Se:Hg ratio ¼ 6.14) potentially because of a greater amount of [THg] in the kidney cortex (Figures 2 and 3) as well as age-dependent factors. In the human kidney cortex under conditions of low Hg concentration, the Se:Hg molar ratio was as high as 300 (minimum [THg] ¼ 0.005 mg/g [36]) which is greater than what was found in kidney cortex of bearded seals (minimum [THg] ¼ 0.164 mg/g) in the present study. When sufficient amounts of Se are available in the prey, Se:Hg molar ratios >1.0 occur in the kidneys of marine mammals and result in the formation of Hg–Se compounds, which are insoluble and hypothetically less toxic than MeHgþ [21,37,38]. The Se:Hg molar ratios in bearded seal heart, kidney cortex, and kidney medulla
Hg and Se by region in bearded seal heart and kidney
were >1.0 in the present study, indicating an adequate supply of Se (based on a simple molar comparison with THg) even in the presence of oxidative stressors such as Hg, which is consistent with previous findings in liver and kidney [21,39]. We recognize that this molar comparison does lack a functional aspect with respect to sequestration of Hg and any antioxidant potential for protection related to Hg-induced oxidative stress. In particular, we note how this important ratio varies between renal cortex and medulla and thus the need to not generalize about Hg and Se relationships in the whole kidney. CONCLUSION
In bearded seals, liver had the highest mean [THg] followed by kidney, skeletal muscle, and heart. In heart, mean [THg] values were relatively low but varied by region, indicating that future studies may consider using more consistent standardized sampling methods. In kidney, [THg] was higher in the cortex than the medulla and should be considered in future sample designs related to monitoring and adverse effects assessment. Greater [TSe] in the kidney than in the heart may indicate a greater antioxidant defense system because of the redistribution of blood to the heart and greater production of reactive oxygen species in the kidney during ischemia/reperfusion episodes associated with diving as well as a response to Hg. However, there may be an important difference for Se-based protection between the cortex and medulla based on the molar ratios. In all tissues, Se:Hg molar ratios greater than 1.0 can be considered a baseline for normal [TSe] under relatively low [THg]. Acknowledgment—The present study would not have been possible without the willingness of hunters to contribute samples from their seal harvests and the support of their communities, local governments, and tribal councils. We thank A. Bryan, M. Nelson, H. Isernhagen, A. Brenner, A. Hunt, J. Bodle, and T. Ray for assistance in collecting and processing samples. We thank A. Barrera-Garcia for expert advice on oxidative stress in aquatic ecosystems and R. Bentzen and J. McIntyre for input on statistical analysis. Funding for sample collection was provided by the National Oceanographic and Atmospheric Administration (grant no. NA08NMF4390544). Samples were collected under National Marine Fisheries Research permit nos. 358-1787 and 15324 issued to the Alaska Department of Fish and Game.
Environ Toxicol Chem 34, 2015
7. 8. 9. 10. 11. 12.
13. 14.
15. 16. 17. 18.
19. 20. 21. 22.
Data availability—Readers may contact L. Correa ([email protected]) to request data.
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31. Dietz R, Paludan-M€uller P, Agger TH, Nielsen CO. 2014. Cadmium, mercury, zinc and selenium in ringed seals (Phoca hispida) from Greenland and Svalbard. In Heide-Jørgensen MP, Lydersen C, eds, Ringed Seals in the North Atlantic. NAMMCO Scientific Publications, Vol 1. North Atlantic Marine Mammal Commission, Tromsø, Norway, pp 242–272. 32. Kanatous S, DiMichele L. 1999. High aerobic capacities in the skeletal muscles of pinnip eds Adaptations to diving hypoxia. J Appl Physiol 86:1247–1256. 33. Saunders D, Fedde M. 1991. Physical conditioning: Effect on the myoglobin concentration in skeletal and cardiac muscle of bar-headed geese. Comp Biochem Physiol A 100:349–352. 34. Garry D, Kanatous S, Mammen P. 2003. Emerging roles for myoglobin in the heart. Trends Cardiovasc Med 13:111–116.
L. Correa et al. 35. Brookens TJ, O’Hara TM, Taylor RJ, Bratton GR, Harvey JT. 2008. Total mercury body burden in Pacific harbor seal, Phoca vitulina richardii, pups from central California. Mar Pollut Bull 56:27–41. 36. Drasch G, Wanghofer E, Roider G, Strobach S. 1996. Correlation of mercury and selenium in the human kidney. J Trace Elem Med Biol 10:251–254. 37. Wang A, Barber D, Pfeiffer C. 2001. Protective effects of selenium against mercury toxicity in cultured Atlantic spotted dolphin (Stenella plagiodon) renal cells. Arch Environ Contam Toxicol 41:403–409. 38. Dietz R, Riget F, Born EW. 2000. An assessment of selenium to mercury in Greenland marine animals. Sci Total Environ 245:15–24. 39. Van de Ven W, Koeman J, Svenson A. 1979. Mercury and selenium in wild and experimental seals. Chemosphere 539–555.
MERCURY AND SELENIUM CONCENTRATIONS IN SKELETAL MUSCLE, LIVER, AND REGIONS OF THE HEART AND KIDNEY IN BEARDED SEALS FROM ALASKA, USA LUCERO CORREA,*y J. MARGARET CASTELLINI,z LORI T. QUAKENBUSH,x and TODD M. O’HARAyk yDepartment of Chemistry and Biochemistry, University of Alaska Fairbanks, Fairbanks, Alaska, USA zSchool of Fisheries and Ocean Sciences, University of Alaska Fairbanks, Fairbanks, Alaska, USA xAlaska Department of Fish and Game, Fairbanks, Alaska, USA kDepartment of Veterinary Medicine, University of Alaska Fairbanks, Fairbanks, Alaska, USA (Submitted 13 December 2014; Returned for Revision 15 February 2015; Accepted 20 May 2015) Abstract: Mean concentrations of total mercury ([THg]) and selenium ([TSe]) (mass and molar-based) were determined for 5 regions of the heart and 2 regions of the kidney of bearded seals (Erignathus barbatus) harvested in Alaska, USA, in 2010 and 2011. Mean [THg] and [TSe] of bearded seal liver and skeletal muscle tissues were used for intertissular comparison. The Se:Hg molar ratios were used to investigate elemental associations and potential antioxidant protection against Hg toxicosis. Age was an important factor in [THg] and Se:Hg molar ratios in heart and kidney. Small but statistically significant differences in mean [THg] occurred among some of the 5 heart regions (p < 0.05). Mean [THg] was highest in liver, 3.057 mg/g, and lowest in heart left ventricle, 0.017 mg/g. Mean [THg] ranked: liver > kidney cortex > kidney medulla > skeletal muscle > heart left ventricle (p < 0.001). Mean [TSe] was highest in liver, 3.848 mg/g, and lowest in heart left ventricle, 0.632 mg/g. Mean [TSe] ranked: liver > kidney cortex > kidney medulla > skeletal muscle > heart left ventricle (p < 0.001). The Se:Hg molar ratios were significantly greater than 1.0 in all tissues (p < 0.001) and represented baselines for normal [TSe] under relatively low [THg]. Mean Se:Hg molar ratios ranked: heart left ventricle > kidney medulla > kidney cortex (p < 0.001). Environ Toxicol Chem 2015;34:2403–2408. # 2015 SETAC Keywords: Mercury
Selenium
Biomonitoring
Molar ratio
Bearded seal
some forms of Hg in the liver and kidney are well known for humans as well as some other animals, whereas methylated forms of Hg are well-known neurotoxins. Liver and kidney are major sites of inorganic mercury (Hg2þ) accumulation, which in many mammals can result in hepato- or nephrotoxicity, respectively [4,12,13]. Lesions in various regions of the liver and kidney have been associated with [THg] above the toxic threshold (60 mg/g wet wt) for marine mammals [12,14,15]. Some forms of Hg can have interactive effects on heart and kidney function, resulting in hypertension or heart disease [13,16,17]. It has been hypothesized that Hg2þ has the potential to interact with sulfur-containing compounds, such as glutathione (GSH), to induce high blood pressure and increase the risk for hypertension and vascular disease [18,19]. Hypertension is thought to be an indirect impact of Hg on the cardiovascular system [18,19]. In many marine mammal studies, [THg] and total concentrations of Se ([TSe]) are positively correlated [20,21]. It has been suggested that a molar Se:Hg ratio significantly greater than 1.0 in certain tissues (such as liver and kidney) may be beneficial to the formation of insoluble HgSe compounds and detoxifying MeHgþ [21]. Selenium concentrations tend to be higher in some piscivorous marine mammals than in terrestrial mammals. In addition, Se can act directly as an antioxidant or as a component of key antioxidants [22,23]. Because of the potential antioxidant role of Se and its direct interactions with Hg, it is also important to understand the distribution of both [THg] and [TSe] in tissues such as heart and kidney. Distributions of Hg and Se in specific regions of the heart and kidney are not usually reported and can prove beneficial in determining biochemical and histopathological effects of mercury. The potential for Hg distribution to differ by region of the heart and kidney is in part the result of the variation in vascularization and concentration of sulfur-containing
INTRODUCTION
Bearded seals (Erignathus barbatus) are 1 of 3 species of sea ice–associated seals used for food in subsistence-based communities in northern Alaska, USA. Population health and status of bearded seals have been monitored since 1962 by the Alaska Department of Fish and Game in cooperation with the Alaska Native subsistence harvest. As an Arctic species, bearded seals are exposed to contaminants such as perfluorinated pollutants [1], organochlorines, and heavy metals such as Hg [2,3]. Numerous studies have been published and many are ongoing related to Hg and Se in vertebrates of the Arctic [4–7]. In particular, Dietz et al. [4] outlined that Hg concentrations in numerous species (such as polar bears, whales, and seals) across the Arctic exceed various guidelines of concern for adverse effects, and they may be increasing over time. Basu et al. [8,9] specifically addressed some of the potential neurotoxicological outcomes and mechanisms of action of some arctic mammals. Thus, it is very relevant to assess Hg and Se concentrations in ice seals, including bearded seals. These animals are important sentinels for assessing potential adverse effects (suite of potential target tissues) and for biomonitoring over space and time (tissues known to be reliably sampled with easily detected concentrations) as outlined in Dietz et al. [4]. Similar to other piscivorous marine species [5], bearded seals bioaccumulate and biomagnify monomethylmercury (MeHgþ) in tissues such as liver, kidney, and skeletal muscle. In arctic seals, especially older piscivores, liver has higher total Hg concentrations ([THg]) than kidney [10,11]. The toxic effects of * Address correspondence to [email protected] Published online 26 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.3079 2403
2404
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compounds, such as GSH and myoglobin, known to bind to various forms of Hg [24–26]. Heart morphology is relatively similar across mammalian species; however, kidney morphology varies (for example, nonreniculated and multireniculated). Seals have multireniculated kidneys, which contain clusters of cortical and medullary structures within each kidney [27,28]. The present study measured [THg] in different regions (intratissular comparisons) of the heart (each chamber wall and the septum) and kidney (cortex and medulla), and compared these values with [THg] in liver and skeletal muscle tissue (intertissular comparisons) [2,3] to understand the regional distribution of [THg] within and among seal tissues. In addition, we measured [TSe] and Se:Hg molar ratios in heart left ventricle and kidney regions (cortex and medulla) to assess regional distribution and relative potential defense mechanisms of Se against Hg toxicosis. MATERIALS AND METHODS
Sample collection
Heart, kidney, skeletal muscle, and liver samples were collected from bearded seals (age kidney medulla > heart (p < 0.001). In all 3 tissues, [TSe] > [THg] (p < 0.001). Statistically significant differences are indicated by different letters.
studies [2,3,10,31]. Even though [THg] is highest in liver, it is known that in adult seals approximately 90% is in the form of Hg2þ, most of which (approximately 53%) is bound to some form of Se and therefore likely to be biologically inactive [2,10]. Liver [THg] and [TSe] in the present study were slightly lower but within the range ([THg] range ¼ 0.64–20.44 and [Se] range ¼ 0.75–23.20 [2]; [THg] range ¼ 1.92–5.22 and [Se] range, 3.40–6.50 [3]) of previous bearded seal studies. Skeletal muscle of bearded seals had greater [THg] than did heart (Table 1). This may be because skeletal muscle has more myoglobin [32,33], which stores and diffuses oxygen within muscle cells [34]. During diving, seals preferentially direct blood to the heart and brain, which maintains an adequate oxygenated blood supply to the heart during long periods of hypoxia, thus reducing the need for heart muscle to use oxygen stored in myoglobin [23]. Myoglobin’s sulfur-containing amino acid cysteine makes it an ideal molecule for binding MeHgþ. Thus, myoglobin may be the reason that 80% or more of THg in skeletal muscle is in the form of MeHgþ [2,35]. The [THg] in skeletal muscle does not vary with age, making this an ideal tissue for monitoring MeHgþ exposure because it eliminates, or at least limits, age as a confounding factor relative to tissues such as liver and kidney. Skeletal muscle [THg] and [Se] in the present study were within the ranges found in Dehn et al. [2].
Mean [THg] and [TSe] in bearded seal heart tissue have not been previously reported. Although the mean [THg] among the 5 regions of the bearded seal heart were different (Figure 1), this result is not likely to be of toxicological or biological significance because the concentrations were low in all regions. In addition, the molar [TSe] was greater than the molar [THg], indicating a potential protective interaction. However, when sampling the heart to determine [THg], it may be important to standardize where the sample is taken (e.g., sample only left ventricle) to reduce the effects that this small variability may have on spatial (populations) and temporal comparisons. In the kidney, we found higher [THg] in cortex than medulla (Figure 2), which was similar to what is reported for terrestrial animals [13,25]. Transport of Hg to the kidney is mediated by sulfur-containing compounds in the blood, such as GSH, albumin, and cysteine. Glutathione has a higher absorption rate into the proximal tubules of the cortex, which likely facilitates the uptake and subsequent accumulation of Hg in the kidney cortex [25]. The disparity between concentrations in the cortex and medulla in the multireniculated kidney suggests that standardized protocols for sampling marine mammal kidneys may be needed to reduce sample variability. Such a protocol might include combining an equal amount of cortex and medulla for analysis, analyzing only the cortex or medulla, or analyzing both the cortex and medulla separately. Sampling cortex and medulla separately may be a problem if a study is focused on young seals because they have smaller renicules, making it difficult to distinguish and dissect cortex and medulla. In adults, however, specific subsampling of cortex and medulla is fairly easy. Thus, the objective of a particular study will dictate the sample protocol that is most appropriate. That [THg] varies with age in the cortex but not in the medulla could be useful in some study designs. For normal biomonitoring purposes, however, it may be best to sample equal proportions of cortex and medulla in animals of a known age to compare [THg] across age groups and for improving temporal and spatial analyses of [THg] in seal monitoring efforts. Kidney [THg] and [TSe] are generally not reported for both cortex and medulla but rather a subsample of the whole kidney. In the present study, analyzing kidney [THg] and [TSe] for cortex and medulla was not different from using whole kidney for comparisons with muscle and heart [2]. Se:Hg molar ratios
Figure 3. Mean ( standard deviation) Se:Hg molar ratio in heart left ventricle, kidney cortex, and kidney medulla tissue of bearded seals. Statistically significant differences among tissues were as follows: heart > kidney medulla > kidney cortex (p < 0.001), as indicated by different letters.
Differences in relative distribution of [TSe] and [THg] (Figure 2) between kidney medulla and heart result in differences in mean Se:Hg molar ratios within these tissues. Mean Se:Hg molar ratios in heart left ventricle (skeletal muscle mean Se:Hg ratio ¼ 50.04) and kidney medulla were greater than that in kidney cortex (liver mean Se:Hg ratio ¼ 6.14) potentially because of a greater amount of [THg] in the kidney cortex (Figures 2 and 3) as well as age-dependent factors. In the human kidney cortex under conditions of low Hg concentration, the Se:Hg molar ratio was as high as 300 (minimum [THg] ¼ 0.005 mg/g [36]) which is greater than what was found in kidney cortex of bearded seals (minimum [THg] ¼ 0.164 mg/g) in the present study. When sufficient amounts of Se are available in the prey, Se:Hg molar ratios >1.0 occur in the kidneys of marine mammals and result in the formation of Hg–Se compounds, which are insoluble and hypothetically less toxic than MeHgþ [21,37,38]. The Se:Hg molar ratios in bearded seal heart, kidney cortex, and kidney medulla
Hg and Se by region in bearded seal heart and kidney
were >1.0 in the present study, indicating an adequate supply of Se (based on a simple molar comparison with THg) even in the presence of oxidative stressors such as Hg, which is consistent with previous findings in liver and kidney [21,39]. We recognize that this molar comparison does lack a functional aspect with respect to sequestration of Hg and any antioxidant potential for protection related to Hg-induced oxidative stress. In particular, we note how this important ratio varies between renal cortex and medulla and thus the need to not generalize about Hg and Se relationships in the whole kidney. CONCLUSION
In bearded seals, liver had the highest mean [THg] followed by kidney, skeletal muscle, and heart. In heart, mean [THg] values were relatively low but varied by region, indicating that future studies may consider using more consistent standardized sampling methods. In kidney, [THg] was higher in the cortex than the medulla and should be considered in future sample designs related to monitoring and adverse effects assessment. Greater [TSe] in the kidney than in the heart may indicate a greater antioxidant defense system because of the redistribution of blood to the heart and greater production of reactive oxygen species in the kidney during ischemia/reperfusion episodes associated with diving as well as a response to Hg. However, there may be an important difference for Se-based protection between the cortex and medulla based on the molar ratios. In all tissues, Se:Hg molar ratios greater than 1.0 can be considered a baseline for normal [TSe] under relatively low [THg]. Acknowledgment—The present study would not have been possible without the willingness of hunters to contribute samples from their seal harvests and the support of their communities, local governments, and tribal councils. We thank A. Bryan, M. Nelson, H. Isernhagen, A. Brenner, A. Hunt, J. Bodle, and T. Ray for assistance in collecting and processing samples. We thank A. Barrera-Garcia for expert advice on oxidative stress in aquatic ecosystems and R. Bentzen and J. McIntyre for input on statistical analysis. Funding for sample collection was provided by the National Oceanographic and Atmospheric Administration (grant no. NA08NMF4390544). Samples were collected under National Marine Fisheries Research permit nos. 358-1787 and 15324 issued to the Alaska Department of Fish and Game.
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7. 8. 9. 10. 11. 12.
13. 14.
15. 16. 17. 18.
19. 20. 21. 22.
Data availability—Readers may contact L. Correa ([email protected]) to request data.
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