fasting period in ringed seals (Pusa [Phoca] hispida) from Svalbard

Comparative Biochemistry and Physiology, Part A 155 (2010) 70–76

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Hormone, vitamin and contaminant status during the moulting/fasting period in ringed seals (Pusa [Phoca] hispida) from Svalbard Heli Routti a,b,⁎, Bjørn Munro Jenssen c, Christian Lydersen a, Christina Bäckman d, Augustine Arukwe c, Madeleine Nyman e, Kit M. Kovacs a, Geir Wing Gabrielsen a a

Norwegian Polar Institute, Polar Environmental Centre, 9296 Tromsø, Norway Centre of Excellence in Evolutionary Genetics and Physiology, Department of Biology, University of Turku, 20014 Turku, Finland Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway d Finnish Food Safety Authority, 00790 Helsinki, Finland e Metsähallitus, Natural Heritage Services, 20300 Turku, Finland b c

a r t i c l e

i n f o

Article history: Received 24 June 2009 Received in revised form 23 September 2009 Accepted 23 September 2009 Available online 2 October 2009 Keywords: Biomarkers Thyroid hormone Vitamin A Vitamin E Calcitriol Thyroid hormone receptor Deiodinase Retinoic acid receptor

a b s t r a c t This study investigates the potential effects of moulting, and the concomitant period of fasting undertaken by ringed seals, on hormone, vitamin and contaminant status in adult animals in a population from Svalbard, Norway, which has relatively low contaminant levels. Concentrations of circulating total and free thyroxine and triiodothyronine, circulating and hepatic vitamin A, hepatic persistent organic pollutants and their circulating hydroxyl metabolites were higher in moulting seals compared to pre-moulting seals. The opposite trend was observed for body condition, circulating calcitriol levels and hepatic mRNA expression of thyroid hormone receptor β. No differences were observed for circulating or hepatic vitamin E levels or hepatic mRNA expressions for deioidinase 1 or 2, or retinoic acid receptor α between the two seal groups. The observed differences are likely the result of increased metabolic rates required during moulting to maintain thermal balance and replace the pelage, in combination with mobilization of lipid soluble compounds from blubber stores during the fasting period that is associated with moulting. The present study shows that contaminant levels and their relationships with physiological or endogenous variables can be highly confounded by moulting/fasting status. Thus, moulting status and body condition should be taken into consideration when using variables related to thyroid, calcium or vitamin A homeostasis as biomarkers for contaminant effects. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Species at the highest levels of marine food chains, such as many marine mammal species, accumulate high levels of persistent organic pollutants (POPs) through their diet. POPs are mainly lipid soluble compounds, which are stored in the blubber. Their toxicokinetics can be strongly influenced by changes in body lipid content (Lydersen et al., 2002; Debier et al., 2003, 2006; Hall et al., 2008). Most Arctic mammals go through a seasonal period of fasting when lipid stores are metabolized and this often leads to mobilization of POPs (Bowen et al., 1987; Atkinson and Ramsay, 1995; Atkinson et al., 1996). Studies have shown that in phocid seals 85–99% of the energy costs incurred during fasting originate from fat catabolism (Nordøy and Blix, 1985; Bennett et al., 2003, Noren et al., 2003). Thus, fasting leads to mobilization of POPs and levels in the blood increase (Lydersen

⁎ Corresponding author. Norwegian Polar Institute, Polar Environmental Centre, 9296 Tromsø, Norway. Tel.: +47 77750500; fax: +47 77750501. E-mail address: [email protected] (H. Routti). 1095-6433/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2009.09.024

et al., 2002). POPs circulating in the blood come into contact with other body compartments, which increases the animal's susceptibility for adverse health effects. Mobilized POPs can induce xenobioticmetabolizing enzymes (e.g. cytochrome P450, i.e. CYP) in the liver during periods of reduced food intake (Wolkers et al., 2008) leading to the formation of highly toxic hydroxyl (OH) metabolites of POPs (Letcher et al., 2000). Thus, fasting periods are believed to be the most susceptible times for contaminant-mediated effects. In marine mammals, various adverse health effects have been associated with high levels of POPs (Ross, 2000; Nyman et al., 2003; Jenssen, 2006). Contaminant effects in free-ranging animals have been studied using biomarkers. Biomarkers are defined as measurable changes in biochemical processes or (endogen) compounds induced by xenobiotics and are therefore indicative of potential pollutionmediated effects (Peakall, 1992). However, biomarkers are regulated in endogenous physiological processes and their concentrations may often vary greatly depending on the life history state of the animal. Therefore, it is important to assess natural variation within potential biomarkers in species; particularly among animals or life-stages that are likely to be exposed to high levels of contaminants.

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Thyroid hormones (THs) have been used as biomarkers to study the effects of POPs in numerous studies in marine mammals (Rolland, 2000; Sormo et al., 2005; Tabuchi et al., 2006; Jenssen, 2006; Hall and Thomas, 2007; Routti et al., 2008a). Recently, gene expression of thyroid hormone receptor has also been suggested as a potential biomarker for contaminant-mediated effects in wildlife (Tabuchi et al., 2006). Thyroid hormones (THs: thyroxine [T4] and triiodothyronine [T3]) play a major role in controlling growth and metabolism, and their levels can be affected by several biological factors including status of development and nutrition (McNabb, 1992). THs are also involved in energetic adaptations to fasting and to moulting and re-growth of the fur in phocid seals (John et al., 1987; Boily, 1996). Thyroid hormones are synthesized in the thyroid gland and T4 is bioactivated to T3 by deiodinases (DIO1 and DIO2) in the peripheral tissues (Gereben et al., 2008). THs are transported in the plasma by carrier proteins and they act via thyroid hormone receptors (TRα and TRβ) (McNabb, 1992). Metabolic rate, and both moulting and fur re-growth are factors that are linked to thyroid hormone levels in seals (Boily, 1996; Kumagai et al., 2006). Seasonal mobilization of lipids from the insulating blubber layer occurs in association with the annual moult, which has impacts on thermoregulatory capacity and metabolic rate. It is therefore likely that these processes might influence the suitability of THs as biomarkers for physiological effects of POPs in seals. Altered levels of calcitriol, a metabolite of vitamin D controlling body calcium homeostasis, have also been linked to exposure to POPs in marine mammals (Routti et al., 2008a). Since serum calcium levels are also related to fasting status in marine mammals (Tryland et al., 2006), it is possible that calcitriol levels might be altered during the fasting period. In phocid seals, moulting coincides with a fasting period (Ryg et al., 1990). It is therefore possible that calcitriol levels vary as a function of the moulting status of seals; this must be taken into account when interpreting calcitriol levels as a biomarker of physiological effects of POPs. Lipid soluble vitamins, such as vitamins A and E, have also been used as biomarkers to study POP-mediated effects in wildlife (Rolland, 2000; Jenssen et al., 2003; Nyman et al., 2003; Debier et al., 2005; Murvoll et al., 2005; Mos et al., 2007; Novák et al., 2008). Vitamin A and its metabolites are defined as “dietary hormones” that regulate the development and physiological homeostasis of various systems (Simms and Ross, 2000; Novák et al., 2008). The most active forms of vitamin A (retinoic acids) act through the retinoic acid receptor (RAR) and retinoic X receptor. Vitamin E is a strong anti-oxidant and is also important for growth and development in animals (Debier and Larondelle, 2005). Because blubber is one of the main storage tissues for vitamins A and E in marine mammals (Schweigert et al., 2002; Mos and Ross, 2002; Debier and Larondelle, 2005), homeostasis of lipid soluble vitamins is likely to be strongly influenced by lipid metabolism during fasting (Schweigert et al., 2002). The ringed seal (Pusa or Phoca hispida) has been recommended as a model species for studying contaminant effects in arctic marine mammals (Arctic Monitoring and Assessment Program (AMAP), 1999). This species has a circumpolar distribution in the Arctic. Ringed seals reproduce in late spring and moult in early summer (March/April and May/June). During these periods their energy requirements, energy consumption and food intake vary widely (Ryg and Oritsland, 1991), and they are generally in a state of negative energy balance, losing 30–35% of their blubber stores (Ryg et al., 1990). The Svalbard ringed seal population is considered to be a healthy population that has low contaminant exposure compared to industrialized areas (Nyman et al., 2002) and minimal hunting pressure; it is thus an interesting reference population for studying ringed seals experiencing other circumstances (Krafft et al., 2006; Tryland et al., 2006). The aim of the present study was to assess the effects of moulting and fasting on thyroid hormone, calcitriol, vitamins A and E and

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contaminant status in ringed seals. Specifically, body condition, plasma concentrations of total and free T4 (TT4, FT4) and T3 (TT3, FT3) and calcitriol, plasma and hepatic concentrations of vitamins A and E, hepatic mRNA expressions for TRβ, DIO1, DIO2 and RARα and catalytic activity of CYP1A, hepatic concentrations of POPs and plasma concentrations of their OH-metabolites were investigated in premoulting and moulting adult ringed seals from Svalbard. We chose to use liver and plasma in this study, because the liver is important in facilitating the metabolism of hormones, vitamins and contaminants, and compounds circulating in plasma are bioavailable for other tissues. 2. Materials and methods 2.1. Field sampling Ringed seals of both sexes from the west coast of Svalbard were sampled in May and June 1996 and 2007 (Table 1). The seals were shot on the sea ice under permits granted to the Norwegian Polar Institute by the Governor of Svalbard or during the local hunting season. The seals were divided into two categories according to their moulting status, pre-moulting and moulting seals. Moulting seals had visible shedding of the hair, whereas the hair was still fast in the premoulting seals (Table 1). The animals were aged based on counting of annual layers in thin transverse sections of the canine teeth. The age structure of the samples was similar in the pre-moulting and moulting ringed seals (Table 1; p = 0.10, Wilcoxon rank sum test). A condition index (blubber percent adjusted for body size) was calculated according to Krafft et al. (2006). Blood and liver samples were obtained immediately after the animals were killed, and the blood samples were centrifuged to obtain plasma. The samples were frozen in liquid nitrogen. Liver samples for mRNA expressions were collected into RNase free cryovials using a sterile knife and frozen immediately in liquid nitrogen. Samples for hormone, vitamin and gene expression analysis were stored at −80 °C. Other samples for chemical analyses were stored at − 20 °C. 2.2. Thyroid hormones and calcitriol Analyses of plasma concentrations of total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), free triiodothyronine (FT3) and calcitriol (1,25-(OH)2-vitamin D) were conducted using radioimmunoassays. TH analyses were performed on samples from both 1996 and 2007, while 1,25-(OH)2-vitamin D analyses were performed only on samples from 2007. Assays for THs were purchased from Siemens Medical Solution Diagnostic (Coat-A-Count, Los Angeles, CA, USA) and that for calcitriol from Diasorin (Sundbyberg, Sweden). A gamma-scintillation counter (Cobra Autogamma Counting System, Model 5003, Packard Instrument, Dowers Grove, IL, USA) was used to register the radioactivity levels of the samples. TT3 and TT4 were analyzed in two to three parallel sample runs, FT4 and FT3 in three parallels, and calcitriol in two parallels. Possible outliers were removed from samples run in series with three parallel runs. Variance between the parallels was 6.4% [4.7, 8.2] for TT4, 3.9% [3.1, 4.8] for TT3, 6.2% [5.0, 7.4] for FT4, 12% [6.1, 17] for FT3, and 8.1% [4.6, 11.7] for calcitriol. Inter- and intra-assay variances were 4.8% for TT4, 6.4% for

Table 1 Number of sampled ringed seals (males and females) from Svalbard. Pre-moulting

Moulting

Year

Males

Females

Age

Males

Females

Age

1996 2007

0 6

1 0

6 9 (6–15)

4 5

4 6

11 (6–17) 13 (5–31)

Geometric mean and range (years) are given for age.

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TT3 and 7.7 % for FT4 and 9.5% for FT3. Intra-assay variance for calcitriol was 6.6%. Because of the high inter-assay variance for calcitriol (38%), the sample concentrations were adjusted according to laboratory controls. Detection limit was 0.94–1.9 nmol/L for TT4, 0.012–0.025 nmol/L for TT3, 0.023–0.11 pmol/L for FT4, 0.092– 0.065 pmol/L for FT3 and 0.96 pg/mL for calcitriol. FT3 levels for two pre-moulting individuals were below the detection limit. These values were replaced by half of the detection limit.

2.3. Vitamins A and E The seal liver samples collected in 2007 were homogenized using a blender. One gram of liver, or plasma, was accurately weighed and then transferred to a 2 mL volumetric flask, which was subsequently filled with Tris–HCl buffer (pH 7.5) solution to the mark. Liver homogenates (0.5 g) were vortexed with 200 μL methanol, 400 μL diisopropyl ether and 40 μL retinyl acetate (5 ng/mL) as an internal standard. After extraction, the ether phase was removed and the sample residue was extracted twice with di-isopropyl ether. The ether phases were combined, filtered over a 0.45 μm filter and evaporated to dryness and finally dissolved into 200 μL of methanol and stored at −70 °C. A 5 μL aliquot was injected into the HPLC system. Internal control samples were prepared (beef liver) in a manner similar to the seal samples. Control samples were stored at −70 °C. A control sample was analyzed with each sample set. Chromatographic conditions were chosen to obtain a good separation of retinoids and α-tocopherol, which was the only tocopherol detected. The experiments were carried out using an HPLC system consisting of a Waters 717 + autosampler, a Waters Alliance™ gradient pump, two detectors, a Waters 996™ photodiode detector (PDA) and a Waters 474 scanning fluorescence detector coupled in series (Waters Inc., Milford, MA, USA). Both retinoids and α-tocopherol were separated on a Grace Vydac (Grace, Deerfield, USA) C18 column (201TP5215, 150 × 2.1 mm, 5 μm) equipped with a corresponding guard column. Retinol and retinyl esters were quantified at 325 nm on the UV detector and α-tocopherol at 292 nm (excitation) and 325 nm (emission) on the PDA detector. The flow rate was set to 0.3 mL/min. The chromatography was carried out using a gradient elution. The solvent system consisted of solvent A (methanol) and solvent B (methanol–water containing10 mM ammonium acetate). The gradient settings were as follows: 0 min 90% B; 0–5 min 45% B; 5–15 min 10% B following by an isocratic elution from15–45 min. Before use, the mobile phases were filtered through a 0.22 μm nylon filter. The chromatographic experiments were carried out at 40 °C. Retinol, retinyl palmitate (16:0), -linoleate (18:2), -oleate (18:1), and -stearate (18:0) were identified by comparison to external standards and confirmed according to the absorption spectra obtained by the photo diode array detector. Retinol and retinol esters were quantified (using retinyl acetate as the internal standard) and retinol equivalents (RE) were calculated. Results for total retinol were expressed as microgram retinol equivalents (RE)/g liver and conversion factors for the individual retinyl esters were calculated as the ratio of the molecular weights of the retinyl ester and retinol (Ross, 1981). The methods used enabled simultaneous detection of retinol and retinyl esters with a good separation of retinyl palmitate and oleate. The reproducibility of the method was b6% and repeatability b20%, calculated as total retinol. The linearity was estimated from an eightpoint calibration of retinol, retinyl palmitate and retinyl acetate using the residual calculation according to NMKL procedure no 4 (Nordic Committee on Food Analysis, 2003). The method was linear within the measuring range from 0.3 to 10 μg/mL. The correlation coefficient was b0.999 for the retinoids. The detection limits were 0.45, 0.02 and 0.04 RE μg/g for retinol, retinyl acetate and retinyl palmitate, respectively. The uncertainty of measurement was 6%, estimated within the laboratory using repeatability of the control samples.

α-tocopherol was quantified according to an external standard method against five concentration levels. The detection limit was 2 μg/100 g and the recovery was 91%. The uncertainty of measurement was 4%, estimated within the laboratory using repeatability of the control samples. 2.4. Quantitative (real-time) PCR Messenger RNA (mRNA) expressions of DIO1, DIO2, TRα and RARα were analyzed in the liver samples collected in 2007. Total RNA was isolated using the TRIzol® Reagent (Invitrogen, Paisley, UK) according to manufacturer's suggested protocol. Purity, stability and concentration of RNA samples were tested using spectrophotometry (NanoDrop Technologies, Wilmington, DE, USA), agarose gel electrophoresis and a combination of poly-T and random primers from an iScript cDNA synthesis kit (used according to the manufacturer's (Bio-Rad Laboratories, Hercules, CA, USA) instructions to generate total cDNA for quantitative PCR (qPCR) using 1 μg of total RNA). High quality RNA with an A260/A280 ratio above 1.9 and intact ribosomal 28S and 18S RNA bands was used for cDNA synthesis. Primers given in 5´–3´ direction (DIO1: Forward-GTGGGCAAAGTGTTCCTGAT, Reverse-GGA CCTTCAGGACAAACCAG (145 bp); DIO2: Forward-GTGGCTGACTTC CTGTTGGT, Reverse-CCCTTTCCTCCCAGATAAGC (101 bp); RARα: Forward-CAGTACTGCCGGCTGCAGAA, Reverse-TGTAGCTCTCGGAGCAC TCG (115 bp); TRβ: Forward-AGAGGCTGGCAAAGAGGA, Reverse-TT TTGATGAGCTCCCACTCC (120 bp)) were designed based on seal and mammalian gene sequences in GenBank and were tested using cDNA pools generated from seal liver from Svalbard and from the Baltic Sea. qPCR was used for evaluating gene expression profiles. For each sample, the expression of individual gene targets was analyzed using the Mx3000P Real-Time PCR System (Stratagene, La Jolla, CA, USA). Each 25-μL DNA amplification reaction contained 12.5 μL of iTAQ™SYBR Green Supermix with ROX (Bio-Rad), 1 µL of cDNA, and 200 nm of each forward and reverse primer. The three-step real-time PCR program included an enzyme activation step at 95 °C for 3 min and 40 cycles at 95 °C (15 s), 57 °C (30 s) and 72 °C (30 s). Controls lacking a cDNA template were included to determine the specificity of target cDNA amplification. Cycle threshold (Ct) values obtained were converted into mRNA copy numbers using standard plots of Ct versus log copy number. The criterion for using the standard curve is based on equal amplification efficiency (usually N90%) with unknown samples and this was checked prior to extrapolating unknown samples to the standard curve. The standard plots were generated for each target sequence using known amounts of plasmid containing the seal amplicon of interest that was cloned and sequenced, as described previously (Mortensen and Arukwe, 2007). All gene expressions were normalized and validated for the quality of the experimental RNA samples using Alien Reference RNA from Stratagene. Data obtained from the individual runs for target cDNA amplification were averaged and expressed as fold changes relative to the alien reference RNA. 2.5. Enzyme assays The activity of CYP1A in the liver microsomes of the seals sampled in 2007 was studied using ethoxyresorufin-O-deethylase (EROD, E.C. 1.14.14.1) activity assay as described elsewhere (Routti et al., 2008b). Results regarding CYP1A activity have been published previously elsewhere (Routti et al., 2008b), but are used herein to study the specific effect of moulting on changes in hepatic CYP1A activity. 2.6. Chemical analysis POPs and their OH-metabolites were analyzed for all samples collected in 2007. The analytical procedures including extraction, partitioning and clean-up, quantification, QA/QC used for the

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determination of hepatic parent compounds and circulating OHmetabolites of polychlorinated biphenyls (PCBs), organochlorine pesticides and polybrominated diphenyl ethers (PBDEs) have been described elsewhere (Routti et al., 2008b, 2009a,b). The contaminant concentration results have been published previously elsewhere (Routti et al., 2008b, 2009a,b), but are used herein to study the specific effect of moulting on changes in their concentrations. Σ-POPs in the liver refers to the sum of Σ43-PCBs, p,p′-DDE, Σ6-cholrdanes, Σ8toxaphenes, Σ6-PBDEs and HCB. Circulating Σ-OH-POPs refers to Σ4OH-PCBs, pentachlorophenol, 4-OH-heptachlorostyrene and 6-OHBDE47. 2.7. Data analysis Statistical analyses were carried out using R version 2.9.0 The differences between pre-moulting and moulting seals were tested using Wilcoxon rank sum test. The differences between pre-moulting and moulting ringed seals in TH concentrations were tested using the adult seals sampled in 1996 and 2007. The effects of moulting on calcitriol, vitamins A and E, gene expressions, condition index, Σ-POPs and their OH-metabolites, and EROD activity were tested in adult seals sampled in 2007, since these parameters were not available for individuals from 1996. Because of the small sample sizes, males and females were combined for the statistical analysis, although sex distribution was not equal between the pre-moulting and moulting seals (Table 1). However, excluding the females did not result in substantial changes in the differences between the pre-moulting and moulting seals. 3. Results Moulting status had a significant effect on the levels of most of the hormones, vitamins and contaminants measured. Mean concentrations of total and free THs, TT4, TT3, FT4 and FT3 were 1.6–3.3 times higher in the moulting seals compared to the pre-moulting seals (Fig. 1). Furthermore, the ratio of FT3:TT3 was higher in the moulting seals. Similarly, but somewhat less prominently, there was a decrease observed in calcitriol levels in the moulting animals (Fig. 1). The mean vitamin A level found in the liver was 3.6 times higher in the moulting seals in comparison with the pre-moulting seals (Fig. 2). Similarly, plasma vitamin A levels were also higher in the moulting animals (Fig. 2). Liver vitamin A was composed mainly of A1 forms of retinyl palmitate, retinol and retinyl oleate, while in plasma only

Fig. 2. Concentrations of total vitamins A and E in the liver and plasma in premoulting and moulting adult ringed seals from Svalbard. Level of significance is expressed as p-value (Wilcoxon rank sum test).

retinol A1 was detected. No difference was observed for vitamin E concentrations in the liver or plasma between the moulting and premoulting seals (Fig. 2). Expressions of hepatic mRNA of TRβ were lower in the moulting seals compared to the pre-moulting seals (Fig. 3). No differences were observed between hepatic expressions of DIO1, DIO2 or RARα mRNA between the moulting and pre-moulting seals. The mean condition index, which reflects whole-body blubber percent adjusted for body size (Krafft et al., 2006), was lower in the moulting compared to the pre-moulting seals (Fig. 4). Mean concentrations for hepatic Σ-POPs and plasma Σ-OH-POPs were 3.3 times and 3.2 times higher, respectively, in the moulting seals compared to the pre-moulting seals (Fig. 4). There was no statistical difference in EROD activity between the pre-moulting and moulting seals (Fig. 4).

4. Discussion The present study compared the status of hormone and vitamin homeostasis and contaminant levels, and explored biotransformation

Fig. 1. Concentrations and ratios of circulating total and free thyroxine (TT4 and FT4, respectively) and triiodothyronine (TT3 and FT3, respectively) and calcitriol in pre-moulting and moulting adult ringed seals from Svalbard. Level of significance is expressed as p-value (Wilcoxon rank sum test).

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Fig. 3. Hepatic mRNA expressions of deiodinases 1 and 2 (DIO1 and DIO2), thyroid hormone receptor (TRβ) and retinoic acid receptor (RARα) expressed as fold change relative to the alien reference RNA. Level of significance is shown as p-value (Wilcoxon rank sum test).

in moulting and pre-moulting ringed seals from Svalbard. The condition index of the seals showed that their condition ranged from low to medium (Tryland et al., 2006). Moulting seals had a lower condition than the pre-moulting seals. None of the seals had a high condition index (Tryland et al., 2006). Although this study is based on quite small sample sizes, it does suggest that moulting status/body condition affects thyroid hormone, calcitriol, vitamin A and contaminant status in ringed seals from Svalbard. The main function of THs is to control metabolism, growth and development from the cellular level to the organismal level (McNabb, 1992). In the present study, concentrations of TH were higher in moulting compared to pre-moulting ringed seals. Interestingly, the bioavailable form of the active TH, FT3, was over three times higher in moulting seals than in pre-moulting seals. The condition index, which reflects the insulating capacity of the blubber layer, was lower in moulting seals than in pre-moulting animals. Because THs play a major role in controlling thermogenesis and maintenance of constant body temperature and basal metabolic rate in homeotherms

Fig. 4. Condition index, and concentrations hepatic Σ-POPs (fresh weight), circulating Σ-OH-POPs (fresh weight) and hepatic EROD activity in pre-moulting and moulting adult ringed seals from Svalbard. Level of significance is shown as p-value (Wilcoxon rank sum test).

(McNabb, 1992), it is possible that the higher levels of THs in the moulting seals are related to thermoregulation and the hair replacement process. In order to accelerate the moulting process phocid seals perfuse their epidermal tissue with blood for weeks (Boily, 1995). Although, the increased blood supply in the epidermis provides nutrients, oxygen and adequate temperature for the growth of new hairs, it also exposes the animals to heat loss, which must be compensated for by increased metabolism (Boily, 1995). In order to minimize heat loss, they prefer to moult on land or ice, since heat flux is much lower in air than in water (Boily, 1995). During moulting, THs stimulate hair growth directly (Ramot et al., 2009) and might also be involved in stimulating the increased metabolism needed for hair regrowth. Elevated TH levels during the moulting period have also been observed in captive grey seals (Halichoerus grypus) (Boily, 1996) and harp seals (Pagophilus groenlandicus) (John et al., 1987). However, in captive harbour seals (Phoca vitulina), moulting status had no effect on THs (Renouf and Brotea, 1991). In grey seals, increased metabolic rate was also observed during the moulting period, and the hormonal and metabolic changes were associated with elevated heat production or the hair re-growth process (Boily, 1996). Elevated resting metabolic rates in moulting captive Steller sea lions (Eumetopias jubatus), which did not decrease their body fat content, suggest that the additional energy produced during moulting is used for hair growth rather than thermoregulation, at least among otariids (Kumagai et al., 2006). Hepatic TRβ mRNA was less expressed in the moulting seals as compared to the pre-moulting seals, which may be a compensatory mechanism for the high levels of bioactive FT3 in plasma in the moulting seals. It is possible that high levels of FT3 in the moulting seals may lead to TH-induced cellular responses primarily in other tissues than the liver, such as in the skin. In general, the present study documents that thyroid homeostasis is influenced by moulting status in ringed seals. Thus, it is important that this is taken into consideration when thyroid-related parameters are applied as biomarkers to assess the physiological effects of POPs in phocid seals. The main function of calcitriol is to regulate calcium homeostasis and bone mineralization. Lower calcitriol levels in moulting seals compared to pre-moulting seals could thus be related to calcium metabolism. Serum calcium levels have been reported to increase with decreasing body condition during the spring in ringed seals from Svalbard (Tryland et al., 2006). Low calcitriol levels in the moulting seals may be linked to reduced calcium uptake by bone (Faibish and Boskey, 2005), which may lead to increased serum calcium levels. Since hair growth has been reported to involve calcitriol among other factors (Ramot et al., 2009), it is also possible that the low calcitriol levels in the moulting seals are related to hair growth. However, it should be noted that moulting was not reported to effect calcitriol levels in southern elephant seals (Mirounga leonina) (Wilske and Arnbom, 1996), though moult in elephant seals is fundamentally different from other phocids. However, the present study suggests that calcitriol levels in ringed seals are influenced by moulting status. Therefore, this factor should be taken into consideration when using calcitriol as a biomarker to assess the contaminant effects in phocid seals. In phocid seals, blubber is the main storage depot for vitamin A (Mos and Ross, 2002; Debier and Larondelle, 2005). Higher levels of vitamin A were found in both the plasma and liver tissues of moulting seals in comparison to the pre-moulting seals. This may be related to vitamin A mobilization from the blubber into other body compartments during fasting. Similar findings have been reported for female grey seals during lactation when they are fasting, which has been attributed to mobilization of vitamin A from the blubber (Debier et al., 2002a; Schweigert et al., 2002). However, the plasma retinol level is normally tightly controlled over a wide range of dietary intakes and liver stores, and it is rapidly regulated by liver uptake or mobilization (Debier and Larondelle, 2005). Therefore, the observed changes in plasma retinol levels could also be related to an altered metabolic rate,

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because retinoic acid, the main bioactive form of vitamin A, enhances whole-body heat production (Bonet et al., 2003). Alternatively, the higher levels of circulating retinol in the moulting seals may play a role in the hair replacement process, because retinoic acid induces hair loss (Ramot et al., 2009), and is involved in the maintenance of the skin (Reichrath et al., 2007). No differences were observed in hepatic or circulating vitamin E concentrations between moulting and pre-moulting seals. In contrast, circulating and hepatic vitamin E levels have been reported to decrease with the onset of lactation in grey seals, which could be explained by the transfer of vitamin E from mothers to pups via the milk (Schweigert et al., 2002). The different responses of vitamins A and E to moulting in the present study of ringed seals suggest that vitamins A and E are not mobilized from storage tissues by similar mechanisms. This finding is in accordance with previous seal studies (Debier et al., 2002a,b; Schweigert et al., 2002). Thus, the present study documents that vitamin A, but not vitamin E, status is influenced by moulting in ringed seals. Thus, vitamin E may be a suitable biomarker that can be used to assess physiological effects of POPs regardless of the moulting status of seals, but that moulting status must be accounted for in the interpretation of vitamin A levels. The higher concentrations of POPs in moulting seals in comparison to pre-moulting animals are almost certainly a result of mobilization of blubber lipids and the POPs contained therein. During moulting the energy balance of seals is negative, resulting to mobilization of blubber lipids. During the moulting period ringed seals spend little time in the water and forage little or not at all. POP mobilization from blubber during fasting has been reported previously in several studies on seals (Lydersen et al., 2002; Debier et al., 2003, 2006; Hall et al., 2008). The increased levels of circulating OH-POPs in moulting versus pre-moulting seals are probably a consequence of increased bioavailability of POPs in the livers of moulting seals, which would induce the activity of xenobiotic enzymes and formation of metabolites. However, hepatic CYP1A activity was not significantly higher in the moulting than in the pre-moulting seals in this study, although negative correlations between the percentage of total blubber and hepatic CYP1A activities have been reported (Wolkers et al., 2008). However, it should be noted that it has been suggested that CYP3A catalyzes the formation of one of the major OH-PCBs detected in these animals (Routti et al., 2008b). It is possible that decreased elimination of OH-metabolites via feces or increased binding sites in plasma due to high TH levels may contribute to the increased levels OH-metabolites in seal plasma during the moult. Thyroid hormones and vitamin levels have been widely used to study potentially adverse health effects of POPs in marine predators (Rolland, 2000; Simms and Ross, 2000; Jenssen et al., 2003; Sormo et al., 2005; Tabuchi et al., 2006; Jenssen, 2006; Mos et al., 2007; Routti et al., 2008a). Both vitamin A and thyroid hormones have been recommended as biomarkers to study the contaminant effects in marine mammals in the Arctic (Arctic Monitoring and Assessment Program (AMAP (1999)). The present study shows that the relationships between contaminants and physiological or endogenous variables may be highly confounded by the moulting status. Thus, it is important to take the moulting status into consideration when using variables related to thyroid, calcium or vitamin A homeostasis as biomarkers. Additionally, the results of the present study confirm that levels of POPs and OH-POPs are directly affected by the moulting status of the animal. Therefore, the timing of sampling with respect to the life history status of phocid seals should be carefully planned in studies investigating contaminant levels and effects. This study did not find any relationship between moulting status and vitamin E levels, so this biomarker may be robust through the moulting season. The present study also demonstrates the importance of having considerable knowledge regarding baseline levels of potential biomarkers when attempting to ascertain the effects of contaminants on animals.

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Acknowledgements Thanks are given to Eero Helle, Jukka Ikonen, Øystein Overrein, Tommy Sandal and Hans Wolkers for their help in sampling in Svalbard. We also thank Christina Lockyer for aging the seals. Martine H. Gjernes, Anne Skjetne Mortensen and Trond M. Kortner kindly provided technical assistance in the gene expression analysis, and Soile Penttinen performed the vitamin analyses. Bert van Bavel and Helen Björnhoft, and Robert Letcher and Shaogang Chu are acknowledged for their contribution to the analysis of POPs and OH-POPs, respectively. This study was financed by Nordic Council of Ministers, the Kone Foundation and the Norwegian Polar Institute. References Arctic Monitoring and Assessment Program (AMAP, 1999. AMAP trends and effects program: 1998–2003 No. 7. Oslo, Norway. Atkinson, S.N., Ramsay, M.A., 1995. The effects of prolonged fasting on the bodycomposition and reproductive success of female polar bears (Ursus maritimus). Funct. Ecol. 9, 559–567. 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