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Mixed function oxidase activity in the harbour seal (Phoca vitulina) from Sable Is., N.S.

One adult male, eight pups (including two full term foetuses) and nine adult female harbour seals (Phoca vitulina) were analysed for indices of mixed function oxidase (MFO) activity. MFO activity was present in liver samples, but was at or below detection limits in samples of kidney, lung and pancreas. Hepatic ethoxyresorufin O-de-ethylase and benzo[a]pyrene hydroxylase activities were similar to those reported in other seals and in other mammals. Cytochromes P-450 and b5 concentrations were slightly lower than those observed in other mammals. MFO activities in newborn pups and foetuses were significantly lower than those in adult females. No qualitative differences in cytochrome P-450 isozyme distribution between foetal and adult samples could be discerned by electrophoresis.

Animals↗

Recent contamination by persistent organochlorines in Baikal seal (Phoca sibirica) from Lake Baikal, Russia.

Blubber samples of Baikal seals pups (Phoca sibirica) collected in 2001 and 2002 were analyzed for persistent organochlorines (OCs) such as PCBs (polychlorinated biphenyls), DDTs (dichlorodiphenyltrichloroethane and its metabolites), HCHs (hexachlorocyclohexane isomers), CHLs (chlordane related compounds), TCPMe [tris(4-chlorophenyl)methane] and TCPMOH [tris(4-chlorophenyl)methanol]. The distribution pattern of OCs was in the following order: DDTs>PCBs>CHLs>HCHs>TCPMOH>TCPMe. Organochlorine concentrations and composition patterns in eight pup blubber samples (2002) analyzed in the present study were compared with the corresponding levels in juvenile Baikal seals from 1992 survey, and it was found that residue levels of OC compounds have declined during the 10 year period. In particular, concentrations of PCBs, HCHs and CHLs in seals collected in 2002 were approximately two times lower, while DDTs levels were three times lower than the corresponding levels in 1992. Furthermore, higher ratios of beta-HCH/HCHs and p,p(')-DDE/DDTs were found in 2002 samples. While comparing the OC levels with those in other seal pups it appeared that all the OC concentrations were lower than the levels in seals from coastal California known as one of the most polluted areas. PCB and DDT concentrations in the present specimens were higher than that in Arctic seal pups. The proportions of tri-, tetra-, penta- and hexachlorobiphenyls in pups captured in 2002 were higher than those in 1992. TEQ concentrations of non- and mono-ortho coplanar PCBs in Baikal seals collected in 2002 were higher than those in seals collected from some locations in the world, where human and industrial activities are extensive, suggesting potential for toxic implications of coplanar PCBs in Baikal seals.

Adipose Tissue↗

Cytochromes P450, P420 & mixed-function oxidases as biomarkers of polychlorinated biphenyl (PCB) exposure in harbour seals (Phoca vitulina).

Hepatic microsomal cytochrome P450, EROD and ECOD activity were investigated as biomarkers of PCB exposure in harbour seals (Phoca vitulina). Due to the difficulty of obtaining undegraded seal liver samples, standard spectrophotometric methodology was adapted to investigate P420 (degraded P450) as a PCB biomarker with partially degraded samples. Total PCB burdens in both blubber and liver had positive correlations with P450, P420 and MFO activity levels. The use of P420 biomarkers in this study supports the inclusion of samples from by-caught marine mammals for future biomonitoring studies. P450 isozymes CYP1A (P4501A) and CYP2B (P4502B) in conjunction with MFO activity were investigated as "specific" biomarkers of PCB exposure. They were found to reliably reflect levels of [MC] and [PB]-type PCB exposure in harbour seal liver.

Adipose Tissue↗

Levels and enantiomeric ratios of alpha-HCH, oxychlordane, and PCB 149 in blubber of harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) from Iceland and further species.

The chiral stationary phase beta-TBDM (35% heptakis(6-O-tert.-butyldimethylsilyl-2,3-di-O-methyl)-beta-cyc lod extrin in OV-1701) was applied to study the enantioenrichment of alpha-HCH, oxychlordane, and PCB 149 in blubber of two seal species from Iceland by gas chromatography with electron capture detection. The examined harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) showed alpha-HCH enantiomeric ratios (ER) > 1. The ER of PCB 149 was comparable in the two species from Iceland but for oxychlordane ER < 1 was observed in harbour seals while the oxychlordane ER in grey seals was > 1. In blubber of Weddell seals (Leptonychotes Weddelli) from the Antarctic we determined an alpha-HCH ER < 1 which is in contrast to the Icelandic seal species and a sample from Lake Baikal.

Animals↗

Levels of organochlorine and polycyclic aromatic compounds in harp seal beaters (Phoca groenlandica).

Chlorinated dibenzo-p-dioxins, dibenzofurans, chlorobiphenyls, organochlorine pesticides and polycyclic aromatic hydrocarbons were measured in blubber, liver, kidney and muscle of five male and five female, 8-month-old harp seals (Phoca groenlandica). Levels of organochlorine contaminants were lowest in muscle, nearly 100 times higher in blubber and intermediate in kidney and liver. Only 2,3,7,8-tetrachlorodibenzofuran was detected in all liver and blubber samples (0.45 and 3.15 pg/g wet wt., respectively). Concentrations of chlorobiphenyls expressed as Aroclor 1254 were 3.8 and 8.2, 8.6 and 21.5, 23 and 73, and 945 and 890 ng/g wet wt. in the muscle, kidney, liver and blubber of female and male seals, respectively. p,p'-DDE, trans-nonachlor, oxychlordane and alpha-HCH were the additional predominant organochlorine compounds at 460 and 460, 415 and 530, 300 and 390, and 190 and 230 ng/g wet in the blubber of female and male seals, respectively. Concentrations of chlorobiphenyls and pesticides in muscle, liver and kidney were two-three times higher in males compared to females, whether expressed on a wet weight or a lipid weight basis, and approximately equal in blubber, even though the blubber layer of females was 30% thicker than that of males. Naphthalene and its C1-C3 homologues were detected in all tissues, generally at concentrations below 10 ng/g wet wt. The concentrations of PCBs and DDTs in blubber are considerably lower than those reported for juvenile seals from the same area 20 years ago. PCB and organochlorine pesticide concentrations and profiles are discussed in relation to those reported from other areas, as well as to sex and age of the seals.

Adipose Tissue↗

Organochlorines in Greenland ringed seals (Phoca hispida).

Twenty-five ringed seals (Phoca hispida) were sampled in 1994 at each of four areas in Greenland, three on the west coast and one on the east coast. Seal blubber samples were analysed for polychlorinated biphenyls (PCBs, IUPAC Nos. 28, 31, 52, 101, 105, 118, 138, 153, 156 and 180), DDTs (p,p'-DDE, p,p'-DDD, p,p'-DDT), hexachlorocyclohexanes (alpha-, beta- and gamma-HCH), hexachlorobenzene (HCB) and trans-nonachlor. A number of the seals (n = 56) were also analysed for toxaphene (total toxaphene and single congeners: CHB Nos. 26, 32, 50 and 62). The overall geometric means (and ranges) found in seals were in microgram kg-1 wet wt. for sigma PCBs: 452 (85-4200), sigma DDTs: 607 (97-6040), sigma HCHs: 123 (51-382), HCB: 13 (3-27), trans-nonachlor: 83 (21-1236) and total toxaphene: 263 (71-950). The geometric means for HCB, trans-nonachlor, toxaphenes and the sums of PCBs and DDTs were higher for the east coast samples than for those from the west coast, and a decreasing trend in concentrations followed the east Greenland current. No geographical trend was apparent for HCHs. Differences in concentrations between females and males were only significant for HCB and HCH within certain age classes and sampling areas. A tendency for concentrations to increase with age was observed, but was not statistically significant. Principal component analyses for PCB congeners revealed that the proportion of higher chlorinated PCBs (Cl atoms > or = 6) decreased with decreasing concentrations of PCBs found for the four sampling areas. No differences between the higher and lower chlorinated PCBs were seen in females and males, but a higher proportion of CB-105 and CB-118 compared to CB-52 and CB-101 was seen in male seals compared to female seals.

Animals↗

Spatial distribution of persistent organochlorines in ringed seal (Phoca hispida) blubber.

Blubber samples, taken through the entire blubber column, were collected from three different anatomical locations on ringed seals (Phoca hispida). The outer and inner layers of these samples were analysed for concentrations of sigma PCBs (sum of the analysed congeners, polychlorinated biphenyls) and p,p'-DDE (4,4'-dichlorodiphenyldichloroethylene). No significant differences were found in blubber thickness or in per cent extractable lipid when comparing age- and sex-groups, or in extractable lipid content when comparing anatomical locations of the blubber or blubber depth. No significant differences were found between the three different anatomical locations with respect to the concentrations of sigma PCBs or p,p'-DDE. However, the concentrations of both sigma PCB and p,p'-DDE were significantly higher in the outer blubber layer compared to the inner. Higher levels of both sigma PCB and p,p'-DDE were found in males compared to females and juveniles in both the inner and outer parts of the blubber column. The most commonly used blubber sampling devices are biopsy tools that penetrate only the outer layer of the blubber. Analyses of such samples will not reflect the real body burden of organochlorines. Standard methods for sampling blubber of marine mammals should be developed so that comparative studies and longitudinal monitoring programs of pollutants in marine mammals can be conducted in a meaningful way.

Adipose Tissue↗

Harbor seal (Phoca vitulina) C-reactive protein (C-RP): purification, characterization of specific monoclonal antibodies and development of an immuno-assay to measure serum C-RP concentrations.

C-reactive protein (C-RP) was purified from harbor seal (Phoca vitulina) serum by calcium dependant phosphoryl-choline and protein A affinity chromatography. Polyacrylamide gel electrophoresis under reducing conditions revealed a single protein moiety with a molecular weight of approximately 25 kDa. An internal peptide derived from this purified protein was subjected to N-terminal amino acid sequencing. A high amino acid sequence similarity was obtained with other published mammalian C-RP molecules confirming that the purified protein was a C-RP homologue. Eight specific monoclonal antibodies (P13, P51, P87, P101, P106, P130, P157 and P219) were raised against this purified protein. All 8 monoclonal antibodies immunoblotted with the 25 kDa C-RP subunit under reducing conditions. A competitive immunoassay was developed identifying elevated C-RP concentrations in harbor seal serum samples with clinical evidence of inflammatory disease. Application of this immunoassay for the measurement C-RP may provide valuable information for the clinical assessment of harbor seal health.

Acute-Phase Reaction↗

cDNA cloning and characterization of an aryl hydrocarbon receptor from the harbor seal (Phoca vitulina): a biomarker of dioxin susceptibility?

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are found at high concentrations in some marine mammals. Species differences in sensitivity to TCDD and PHAHs are a major limitation in assessing the ecological risk to these animals. Harbor seals accumulate high levels of PHAHs and are thought to be highly sensitive to the toxic effects of these compounds. To investigate the mechanistic basis for PHAH toxicity in harbor seals (Phoca vitulina), we sought to characterize the aryl hydrocarbon receptor (AHR), an intracellular protein that is responsible for PHAH effects. Here we report the cDNA cloning and characterization of a harbor seal AHR. The harbor seal AHR cDNA has an open reading frame of 2529 nucleotides that encodes a protein of 843 amino acids with a predicted molecular mass of 94.6 kDa. The harbor seal AHR protein possesses basic helix-loop-helix (bHLH) and Per-ARNT-Sim (PAS) domains. It is most closely related to the beluga AHR (82%) and human AHR (79%) in overall amino acid identity, indicating a high degree of conservation of AHR structure between terrestrial and some marine mammals. The ligand binding properties of the harbor seal AHR were determined using protein synthesized by in vitro transcription and translation from the cloned cDNA. Velocity sedimentation analysis on sucrose gradients showed that the harbor seal AHR exhibits specific binding of [(3)H]TCDD. The [(3)H]TCDD-binding affinity of the harbor seal AHR was compared with that of the AHR from a dioxin-sensitive mouse strain (C57BL/6) using a hydroxylapatite assay. The equilibrium dissociation constants of seal and mouse AHRs were 0.93+/-0.19 and 1.70+/-0.26 nM, respectively. Thus, the harbor seal AHR bound TCDD with an affinity that was at least as high as that of the mouse AHR, suggesting that this seal species may be sensitive to PHAH effects. The characteristics of the AHR potentially can be used as a biomarker of susceptibility to dioxin-like compounds, contributing to the assessment of the risk of these compounds to marine mammals and other protected animals.

Amino Acid Sequence↗

Organochlorine and organotin compounds in Caspian seals (Phoca caspica) collected during an unusual mortality event in the Caspian Sea in 2000.

Polychlorinated biphenyls (PCBs), organochlorine pesticides and organotin compounds were determined in the blubber and liver of Caspian seals (Phoca caspica) found stranded on the coast of the Caspian Sea during an outbreak of canine distemper virus (CDV) in 2000. Among organochlorines analyzed, DDTs were the most dominant contaminants with concentrations ranging from 6.3 to 470 microg/g on a lipid-weight basis. Caspian seals collected in 2000 during the epizootic had higher concentrations of organochlorines than healthy individuals sampled in 1998. However, the blubber layer was generally thinner in the seals collected in 2000 than those in the previous surveys. Although compositions of organochlorine pesticides in seals suggested that the contamination status in the Caspian Sea is improving, the levels found in Caspian seals in 2000 were comparable to those in other marine mammals that have suffered from epizootics. This implies that the present status of contamination found in Caspian seals poses a risk of immunosuppression. Concentrations of butyltin compounds in livers of seals ranged from 0.49 to 17 ng/g on a wet-weight basis and octyltin compounds were below limit of detection in all the samples analyzed, suggesting less contamination by organotin compounds in the Caspian Sea.

Algorithms↗

Current levels of DDT, PCB and trace elements in the Baltic ringed seals (Phoca hispida baltica) and grey seals (Halichoerus grypus).

Residue levels of polychlorinated biphenyls (PCBs) and 1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane (DDT) were determined from liver samples of ringed seals (Phoca lispida) from the Baltic Sea and Svalbard, and of grey seals (Halichoerus grypus) from the Baltic Sea and Sable Island in Canada. Both Baltic seal populations showed clearly higher average sum PCB (SPCB) and sum DDT (SDDT) levels than the reference seal populations. Among the Baltic seals, SPCB levels were twice as high as SDDT levels, and both contaminants were higher in ringed seals than in grey seals. A difference in gender was observed only in the Sable Island grey seal population, in which males showed a higher level of contamination than females. A reduction of the SDDT levels, and to a lower extent of the SPCB levels can be observed in the Baltic seals since the peak contaminant levels during the 1970s. The decrease has been more rapid in the grey seals than in the ringed seals. The SPCB levels in the Baltic ringed seals are still high enough to cause a threat to their well being. Residue levels of the trace elements mercury, cadmium, lead and selenium were determined from liver, kidney and muscle samples of grey seals from the Baltic Sea and Sable Island. Only cadmium showed a geographic difference being higher in the seals from Sable Island than from the Baltic Sea. No clear reduction of the metal burden can be observed in the Baltic grey seal population since the 1970s.

Age Factors↗

Bioaccumulation profiles of polychlorinated biphenyls including coplanar congeners and possible toxicological implications in Baikal seal (Phoca sibirica).

Isomer specific concentrations of individual polychlorinated biphenyls (PCBs) including toxic non-ortho (IUPAC 77, 126, 169), mono-ortho (105, 118, 156) and di-ortho (137, 138, 153, 180) coplanar congeners were determined in the blubber of 40 Baikal seals (Phoca sibirica) and as their fish diet collected from Lake Baikal, Siberia. Residue levels of total PCBs in Baikal seals were noticeably high and comparable to those reported for seals from the North Sea, suggesting the recent usage of this compound in the watershed of Lake Baikal. Non-, mono-, and di-ortho coplanar congeners were also detected in Baikal seals and fish. An approach to estimate bioaccumulation profiles of PCB congeners revealed that the non-ortho PCBs, IUPAC 77, 126 and 169 seemed to be less persistent than other congeners. Furthermore, selective biotransformation of PCB congeners having either meta-para vicinal H atoms or both adjacent chlorinated meta-para and ortho-meta positions has been suggested. Comparison of 2,3,7,8-TCDD toxic equivalents (TEQ) of non-, mono- and di-ortho coplanar congeners in Baikal seals with those for other marine mammals suggested higher enrichment of mono-ortho congeners, particularly IUPAC 105 and 118, which contributed significantly to the total TEQs in Baikal seals. Results imply that the TCDD-like toxicity is relatively serious in Baikal seals, because of the enrichment of these toxic PCB congeners in tissues.

Journal Article↗

Geographical variation of persistent organochlorine concentrations in blubber of ringed seal (Phoca hispida) from the Canadian Arctic: univariate and multivariate approaches.

Geographical variation of organochlorine (OC) concentrations in ringed seal (Phoca hispida) in the Canadian Arctic was studied using univariate and multivariate statistical techniques. The dataset consisted of 80 individual OC components (58 PCB congeners plus DDT- and chlordane (CHL)-related compounds, toxaphene, hexachlorocyclohexanes (HCH), chlorobenzenes (CBz), and dieldrin) determined in 221 ringed seal blubber samples from 13 locations throughout the Canadian Arctic from the period 1983 to 1989. Mean concentrations of the major OC groups in ringed seal blubber (SigmaPCBs, SigmaDDT, SigmaCHL and toxaphene), adjusted for the age and sex of the seals, showed few significant geographical differences. Principal components analysis was used to examine geographical trends. Significant differences in mean factor scores for three of four principal components were found between sampling locations. Locations in the western and high Arctic could be distinguished from those in Hudson Bay by highest scores along principal component two which was associated principally with CBz. PCB congeners with six or more chlorine substitutions declined with increasing north latitude, whereas more volatile OCs (CBz, HCH, less chlorinated PCBs) increased in the proportion of total OCs with latitude. Proportions of less chlorinated PCBs also decreased with increasing longitude, whereas slopes of regressions for more highly chlorinated PCBs increased significantly. The results were generally consistent with the 'cold condensation' hypothesis of increasing proportions of more volatile OCs with increasing latitude and distance from sources.

Journal Article↗

Vitamins A1 (retinol), A2 (3,4-didehydroretinol) and E (alpha-tocopherol) in the liver and blubber of lacustrine and marine ringed seals (Phoca hispida sp.).

Concentrations of vitamins A1 (retinol), A2 (3,4-didehydroretinol) and E (alpha-tocopherol) in the liver and blubber of ringed seals from Lake Saimaa (Phoca hispida saimensis), Lake Ladoga (P. h. ladogensis), the Baltic Sea (P. h. botnica) and Spitsbergen (P. h. hispida) were determined by high-performance liquid chromatography. The freshwater seals had much lower levels of vitamin A1 but higher levels of vitamin A2 than the marine seals. The concentrations of vitamin E in the livers of the subspecies studied were high compared with earlier reports of seals, but the ranges were large. The livers of the marine seals contained more vitamin E than the livers of the freshwater seals, but the levels in the blubber were uniform in all populations, except in old specimens from the Baltic. The differences between the freshwater and marine seals are suggested to be due mainly to diet. The ratios of A1 to A2 in the liver and blubber and in the fish diet were similar for the marine seals and for the freshwater seals (but differed in the marine and freshwater populations), which suggests no great differences in the absorption, transport and metabolism of the two analogues. Blubber was an important storage site for the vitamins studied, and age-dependent increases were detected, especially for vitamin E. In the 2-month to 2-year-old ringed seals of Lake Saimaa, however, the vitamin E concentration in the blubber was not affected by age.

Adipose Tissue↗

Composition of polyunsaturated fatty acids in the liver of freshwater and marine ringed seals (Phoca hispida ssp.) differs largely due to the diet of the seals.

Fatty acid composition in the liver of four ringed-seal (Phoca hispida ssp.) populations, from ocean (Spitsbergen), brackish water (the Baltic Sea) and freshwater (Lake Saimaa and Lake Ladoga) were determined by gas liquid chromatography. The fatty acid compositions in liver were compared with those of the blubber of the seals, which largely reflect the fatty acid supply from marine or freshwater fish. When the ratios of 20:4n-6/20:5n-3 (arachidonic acid/eicosapentaenoic acid, AA/EPA, from 0.06 to 0.55) and n-6 PUFA/n-3 PUFA (n-6/n-3, from 0.09 to 0.35) increased from the marine to freshwater blubber, the corresponding increase in these ratios in the total lipids of the liver was 10-fold or more (AA/EPA: from 1.2 to 13.5 and n-6/n-3: from 0.9 to 3.2). Thus, when available in the diet, AA is preferentially incorporated into the lipids of pinniped liver.

Adipose Tissue↗

Humoral immune responses to phocine herpesvirus-1 in Pacific harbor seals (Phoca vitulina richardsii) during an outbreak of clinical disease.

Infection with phocine herpesvirus type-1 (PHV-1) has been associated with morbidity and high mortality in neonatal harbor seals (Phoca vitulina). A PHV-1 specific indirect enzyme linked immunosorbent assay (ELISA) was developed to sequentially measure the serological status of 106 harbor seal neonates admitted to a Pacific coast rehabilitation center (total number of sera tested was 371). Early in the season (February-April), the majority of pups had low serum levels of PHV-1 specific antibody. A dramatic increase in PHV-1 specific antibody, involving the majority of hospitalized pups, was observed during a 4-week period in May. This coincided with a high incidence of PHV-1 associated adrenal lesions and mortality. Although there was overall agreement between the timing of seroconversion to PHV-1 and histological evidence of PHV-1 infection, 82.4% of individual pups with adrenalitis had no evidence of a humoral response to PHV-1 at the time of their death. This suggests either a rapid disease course, or an inability to develop a humoral response in some neonatal seals.

Animals↗

Chemical characterization of the oligosaccharides in milk of high Arctic harbour seal (Phoca vitulina vitulina).

Carbohydrates were extracted from high Arctic harbour seal milk, Phoca vitulina vitulina (family Phocidae). Free neutral oligosaccharides were separated by gel filtration and preparative thin layer chromatography, while free sialyl oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows: lactose, 2'-fucosyllactose, lacto-N-neotetraose, lacto-N-neohexaose, monofucosyl lacto-N-neohexaose and difucosyl lacto-N-neohexaose. Thus, all of the neutral saccharides contained lactose or lacto-N-neotetraose or lacto-N-neohexaose as core units and/or non-reducing alpha(1-2) linked fucose. These oligosaccharides have also been found in hooded seal milk. The structures of the silalyl oligosaccharides were: monosialyl lacto-N-neohexaose, monosialyl monofucosyl lacto-N-neohexaose, monosialyl difucosyl lacto-N-neohexaose and disialyl lacto-N-neohexaose. These oligosaccharides contained lacto-N-neohexaose as core units, and one or two alpha(2-6) linked Neu5Ac, and/or non-reducing alpha(1-2) linked Fuc. The Neu5Ac residues were found to be linked to GlcNAc or penultimate Gal residues. The acidic oligosaccharides are the first to have been characterized in the milk of any species of seal.

Animals↗

The effect of body fat on basal metabolic rate in adult harp seals (Phoca groenlandica).

Three adult harp seals (Phoca groenlandica) were fed different daily amounts of capelin (Mallotus villosus), and their body composition determined by use of the tritiated water method at different levels of fattening. Basal metabolic rate (BMR) was measured after 5 days of fasting by indirect calorimetry, and was on average 1.1 W.kg-1 when 45% of body mass (BM) was fat and 2.3 W.kg-1 when body fat was reduced to 13% of BM. This suggests that body fat contributes little to BMR in these animals. It follows, that predictions of BMR on the basis of BM is questionable in seals, in which body fat may change seasonally between 20 and 60% of BM.

Adipose Tissue↗