Towards molecular understanding of species differences in dioxin sensitivity: initial characterization of Ah receptor cDNAs in birds and an amphibian

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are highly toxic to most vertebrate animals, but there are dramatic species differences in sensitivity, both within and among vertebrate classes. For example, studies in cultured avian hepatocytes have revealed differential sensitivity of birds to PHAHs [Kennedy et al. (1996). Toxicol. Appl. Pharmacol., 141, 214-230]. Differences in the characteristics or expression of the aryl hydrocarbon receptor (AHR) could contribute to these species differences in PHAH responsiveness. To investigate the molecular mechanism of differential PHAH sensitivity, we have begun to characterize the AHR in white leghorn chicken (Gallus gallus), Pekin duck (Anas platyrhynchos), and common tern (Sterna hirundo), as well as an amphibian, mudpuppy (Necturus maculosus). Partial AHR cDNAs encompassing the helix-loop-helix and PAS domains were cloned and sequenced. Comparison of amino acid sequences in this region indicated a high degree of sequence conservation among the bird species (97% amino acid identity). The percent identity between bird sequences and either mouse or mudpuppy was lower (79%); the mudpuppy AHR was 74% identical to the mouse AHR. Phylogenetic analysis of these and other AHR amino acid sequences showed that the bird and mudpuppy AHRs were more closely related to mammalian and fish AHR1 forms than to fish AHR2. Future studies include the in vitro expression and functional characterization of AHRs from these and other non-mammalian vertebrates.     

Identification of aryl hydrocarbon receptor 2 in aquatic birds; cDNA cloning of AHR1 and AHR2 and characteristics of their amino acid sequences

The toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its related planar halogenated aromatic hydrocarbons (PHAHs) are mediated by the aryl hydrocarbon receptor (AHR). To investigate the potential sensitivity to PHAHs and the evolutional diversity of AHR in aquatic birds, AHR cDNAs were initially cloned and sequenced from the livers of a black-footed albatross (Diomedea nigripes) and a common cormorant (Phalacrocorax carbo). In this study, we report the identification of two distinct AHR paralog genes in these species. The two full-length AHR cDNAs from albatross were highly divergent (33% overall amino acid identity, and 60% identity in the N-terminal half). Phylogenetic analysis showed that one of them belongs to the AHR1 clade and the other one to the AHR2 clade, which has been identified only from fishes, but not yet from mammals and birds. Albatross AHR1 encoded a 861-residue protein with a predicted molecular mass of 96.7 kDa, and in the case of albatross AHR2, 925 amino acids and 100.7 kDa. From cormorant liver, the full-length AHR1 cDNA and the partial AHR2 cDNA were cloned. This result strongly suggests that bird species also possess two distinct AHR genes (AHR1 and AHR2). To our knowledge, this is the first report on the presence of an AHR2-like isoform in bird species as well as in fish.     

斑鳢(Channa maculata)微囊藻毒素去毒相关基因sGST的克隆与序列分析

采用RT-PCR技术和RACE技术成功克隆淡水鱼类斑鳢sGST基因cDNA全序列,推测得到氨基酸序列,初步分析其结构功能域及系统进化关系。结果表明,斑鳢sGST基因cDNA序列全长为898bp,编码225个氨基酸。斑鳢sGST与真鲷、金头鲷、鲽、黑头鲦、川鲽、大口黑鲈等最新定名为ρ型的sGST氨基酸同源性较高,ρ型sGST为水生生物所特有并共同占据进化树上独立的分枝;与大鼠、小鼠、人等哺乳动物sGST现有所有类型同源性均很低,并且在进化树上距离也较远,表明本研究成功克隆的sGST基因应属于ρ型,可能在鱼类等水生生物对水栖环境的适应上有重要作用。     

Cytochrome P450 in seals: monooxygenase activities, immunochemical cross-reactions and response to phenobarbital treatment

Cytochrome P450 monooxygenase activities, reflecting the expression of various subfamilies of P450, were measured in liver samples of harp and hooded seal. Differences in some of the investigated parameters were observed between the two species, between sexes, and between pups and adults. Treatment of single female pups from each species with phenobarbital (i.v. 45 mg/kg), resulted in increased levels of EROD and estradiol 2-hydroxylase activities in both species, whereas MCOD, ECOD and PROD activities were induced only in the harp seal sample. Antibodies against a dog P450 2B form (anti-dog PBD-2 IgG) gave a single band around 52kD in both species, strongest in male pups. This band seemed elevated in the PB-treated harp seal pup. Based on the single treated pup of each species, the results suggest that seals respond to PB-type treatment with a weaker response, and with different enzyme patterns, that most terrestrial mammals. Antibodies against cod P450 IAI cross-reacted with two bands in liver samples from adult seals of both species (about 54 and 52kD), but this was strongest in the hooded seal. The intensity of the bands reflected the EROD activities in the samples, suggesting the application of immunodetection in screening marine mammals for effects of environmental contamination.     

Accumulation of dietary organochlorines and vitamins in Baltic seals

We studied the accumulation of polychlorinated biphenyls (PCBs), 1,1,1.trichloro-2,2-bis[p-chlorophenyl]ethane (DDT) and its metabolites, and vitamins A and E in the top levels of the Baltic Sea food web. The investigation focused on the transfer of contaminants and vitamins to the ringed seal (Phoca hispida baltica) and the grey seal (Halichoerus grypus) from their main prey species. The trophic level of the seals was investigated using stable isotopes of nitrogen and the results indicated that both species of Baltic seal feed at approximately the same level. PCBs accumulated to a greater extent in the grey seal than in the ringed seal. Biomagnification factors for DDT compounds were similar for both species of Baltic seal (85-140). Differences in observed DDT levels were due to different prey selection by the two species, while differences in PCB levels indicated a species-specific metabolic system. There was a clearly greater accumulation of DDT compounds than of PCBs in both species of seal. The supply of dietary vitamin A was normally above the recommended level in all the seal populations studied. Low levels of hepatic vitamin A in the Baltic seals could therefore indicate the toxic effects of a high level of persistent organic pollutants on vitamin A dynamics, at least in the ringed seal. In the grey seals, low hepatic vitamin A levels could also be explained by lower levels of dietary vitamin A, compared to the reference grey seals, as it is not known if seals can store unlimited amounts of vitamin A. The greater uptake of vitamin E by Baltic seals, compared to seals in the reference areas, demonstrated by elevated levels of vitamin E in the blubber, could be a response to oxidative stress caused by the high contaminant load. These results further support our previous hypothesis that the toxic effects of environmental contaminants could be causing the observed divergence in vitamin levels between the Baltic seals and the reference seal populations.     

Prioritising range-wide scientific monitoring of the Cape fur seal in southern Africa

The range of the Cape fur seal Arctocephalus pusillus pusillus population largely coincides with the region of the cold, nutrient-rich Benguela Current Large Marine Ecosystem (BCLME) adjoining the west coast of South Africa, Namibia and Angola. Range-wide scientific monitoring of the seal population was initiated in the 1970s to inform on population management questions related to commercial seal harvesting and seal–fishery interactions. Since the 1970s, seal-related management goals have changed, especially in South Africa where seal harvesting ceased in 1990 and government has conformed to scientific advice against the culling of seals for the intended benefit of fisheries. However, renewed impetus has been provided to seal research and monitoring through the expansion of the ‘ecosystem-based management’ concept in the region, as well as improved international cooperation in the management of marine resources. Together with wide-scale ecosystem changes in the marine environment, and forecast effects of global climate changes, this has justified the continuation and improvement of range-wide scientific monitoring of Cape fur seals. We prioritised seal monitoring based on cost, effort, and relevance to monitoring objectives that have been identified for the region, with consideration given to the conservation status of top predators, interspecific and predator-fishery interactions and the potential use of Cape fur seals as indicators of ecosystem health. An integrated approach incorporating a suite of life-history attributes of seals is recommended, useful monitoring tools are discussed and the need for coordinated monitoring effort and standardisation of sampling techniques is emphasised.     

Polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) in livers of harbor seals (Phoca vitulina) from San Francisco Bay,California and Gulf of Maine

Bioaccumulation of endocrine disruptors in marine mammals positioned at the top of the food chain is of toxicological concern. Livers from four pups and ten adult harbor seals (Phoca vitulina) stranded in San Francisco Bay (SFB) and the Gulf of Maine (GOM) were analyzed for polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs). We used GC–ECD and GC–NCI/MS to investigate the presence of 28 PCBs and 8 OH-PCB metabolites, respectively. Σ₂₈PCB concentrations (di- to octa-CBs) ranged from 1.81 to 35.9 μg/g lipid with a median of 6.53 for the seal pups and 2.31 to 249 μg/g lipid with a median of 28.9 for the adult seals. Σ₈OH-PCB concentrations (penta- to hepta-OH-PCBs) ranged from 0.02 to 0.69 μg/g lipid with a median of 0.04 for the adult seals, i.e., at much lower concentrations than those for PCBs. Ratios of OH-PCBs to PCBs (0.24% on average) were comparable to those in beluga whale, but were lower than ratios in human livers. The OH-PCB profiles were slightly different between SFB and GOM seal livers, although similar PCB congener patterns were observed. Generally, 4-OH-CB107 was found predominantly in seal livers and was the only OH-PCB detectable in most of seal pup livers. This study provides information on OH-PCBs in seals, adding to the scarce exposure data for these chemicals.     

Biological transport and mammal to mammal transfer of organochlorines in Arctic fauna

Ringed seal (Phoca hispida) is assumed to be the most important and common prey of polar bears (Ursus maritimus). However, during a scientific survey in the ice area of the northern Barents Sea east of Svalbard in June 1995, an unexpectedly high number of polar bears were observed feeding on harp seal (Phoca groenlandica) carcasses. Samples of both harp and ringed seals were obtained and organochlorine (OC) occurrence and pattern in these two potential polar bear prey species were determined. Significantly higher OC concentrations were found in harp seals, as compared to the ringed seals. All animals in the northern harp seal group were lean specimens in late moult. The industrial chemicals, polychlorinated biphenyls (PCBs) and hexachlorobenzene (HCB), and the OC pesticides bis-2,2,(chlorophenyl)-1,1,1-trichloroethanes (DDTs), hexachlorocyclohexanes (HCHs), and chlordanes (CHLORs) were analysed in blubber. The concentrations of sigma PCB (sum of concentrations of 16 PCB congeners) and sigma DDT (sum of concentrations of p,p'-DDT and p,p'-DDE) in the northern harp seal group ranged from 2093 to 20,382 and 1460 to 10,381 ng g-1 lipid weight, with mean concentrations of 11,133 and 6847 ng g-1 lipid weight, respectively. The mean concentrations of the CHLORs, oxychlordane and trans-nonachlor, were 1311 and 3743 ng g-1 lipid weight, respectively, while the mean concentrations of HCB and HCH isomers (alpha-, beta- and gamma-HCH) were all < 500 ng g-1 lipid weight. No significant difference was found in the mean total blubber mass between the two seal species when collected in June. This indicates that polar bears preying on harp seals instead of ringed seals at this time of the year could accumulate significantly higher PCB concentrations. We suggest that polar bears feeding along the ice-edge east of Svalbard in May and June preferentially prey on harp seals instead of ringed seals, and that this may partly explain the variation in PCB concentrations among polar bears from the Norwegian Arctic. An hypothesis is that the harp seal may function as a transport vector of OCs into the high Arctic environment.     

Theoretical aspects of cap-rock and fault seals for single- and two-phase hydrocarbon columns

Cap-rock seals can be divided genetically into those that fail by capillary leakage (membrane seals) and those whose capillary entry pressures are so high that seal failure preferentially occurs by fracturing and/or wedging open of faults (hydraulic seals). A given membrane seal can trap a larger oil column than gas column at shallow depths, but below a critical depth (interval), gas is more easily sealed than oil. This critical depth increases with lower API gravity, lower oil GOR and overpressured conditions (for the gas phase). These observations arise from a series of modelling studies of membrane sealing and can be conveniently represented using pressure/ depth (P/D) profiles through sealed hydrocarbon columns. P/D diagrams have been applied to the more complex situation of the membrane sealing of a gas cap underlain by an oil rim; at seal capacity, such a two-phase column will be always greater than if only oil or gas occurs below the seal.These conclusions contrast with those for hydraulic seals where the seal capacity to oil always exceeds that for gas. Moreover, a trapped two-phase column, at hydraulic seal capacity will be less than the maximum-allowed oil-only column, but more than the maximum gas-only column. Unlike membrane seals, hydraulic seal capacity should be directly related to cap-rock thickness, in addition to the magnitude of the minimum effective stress in the sealing layer and the degree of overpressure development in the sequence as a whole.Fault-related seals are effectively analogous to membrane cap-rocks which have been tilted to the angle of the fault plane. Consequently, all of the above conclusions derived for membrane cap-rocks apply to both sealing faults sensu stricto (fault plane itself seals) and juxtaposition faults (hydrocarbon trapped laterally against a juxtaposed sealing unit). The maximum-allowed two-phase column trapped by a sealing fault is greater than for equivalent oil-only and gas-only columns, but less than that predicted for a horizontal membrane cap-rock under similar conditions. Where a two-phase column is present on both sides of a sealing fault (which is at two-phase seal capacity), a deeper oil/water contact (OWC) in one fault block is associated with a deeper gas/oil contact (GOC) compared with the adjacent fault block. If the fault seal is discontinuous in the gas leg, however, the deeper OWC is accompanied by a shallower GOC, whereas a break in the fault seal in the oil leg results in a common OWC in both fault blocks, even though separate GOC's exist. Schematic P/D profiles are provided for each of the above situations from which a series of fundamental equations governing single- and two-phase cap-rock and fault seal capacities can be derived. These relationships may have significant implications for exploration prospect appraisal exercises where more meaningful estimates of differential seal capacities can be made.The membrane sealing theory developed herein assumes that all reservoirs and seals are water-wet and no hydrodynamic flow exists. The conclusions on membrane seal capacity place constraints on the migration efficiency of gas along low-permeabiligy paths at depth where fracturing, wedging open of faults and/or diffusion process may be more important. Contrary to previous assertions, it is speculated that leakage of hydrocarbons through membrane seals occurs in distinct pulses such that the seal is at or near the theoretically calculated seal capacity, once this has been initially attained.Finally, the developed seal theory and P/D profile concepts are applied to a series of development geological problems including the effects of differential depletion, and degree of aquifer support, on sealing fault leakage, and the evaluation of barriers to vertical cross-flow using RFT profiles through depleted reservoirs. It is shown that imbibition processes and dynamic effects related to active cross-flow across such barriers often preclude quantitative analysis and solution of these problems for which simulation studies are usually required.     

A review of animal welfare implications of the Canadian commercial seal hunt

The Canadian seal hunt is the world's largest commercial slaughter of marine mammals and, as such, has been the subject of veterinary scrutiny for half a century. In that time, veterinary experts have made multiple recommendations to improve welfare at the seal hunt, some of which have been included in Canadian sealing regulations. Yet analysis of video material and studies on the outcomes of the hunt suggest that the potential for suffering during the hunt continues, and may, in fact, be increasing. In the past decade, numerous countries have taken action to prohibit their trade in products of commercial seal hunts in response to public concerns about the welfare of the seals. With these actions now being examined at the World Trade Organization, it is important to determine if these concerns are warranted. This paper reviews relevant veterinary science, exploring the intrinsic elements of commercial sealing and unique physical adaptations of seals that prevent effective and consistent application of humane slaughter methods at the seal hunt. The review of available data indicates that generally accepted principles of humane slaughter cannot be carried out effectively or consistently in the commercial seal hunt.     

Assessment of cytochrome P450 1A in harbour seals (Phoca vitulina) using a minimally-invasive biopsy approach

Biomarkers of organochlorine exposure, such as the induction of cytochrome P450 1A (CYP1A), can be used to assess the impact of environmental contaminants on the health of free-ranging marine mammal populations. The objective of the present study was to measure CYP1A in skin and liver biopsies obtained from live harbour seals (Phoca vitulina). Twelve harbour seal pups, aged three to five weeks, were captured from the Fraser River estuary, British Columbia, Canada, and temporarily held in captivity. Skin ( approximately 60 mg) and liver ( approximately 40 mg) biopsies, obtained while seals were under general anaesthesia, yielded sufficient tissue for the measurement of CYP1A by immunoblot analysis and ethoxyresorufin O-deethylase activity. A short-term exposure experiment, in which harbour seals (n=3) were treated orally with beta-naphthoflavone (BNF), resulted in increased hepatic and cutaneous CYP1A protein levels, consistent with observations in other mammals. This study is the first to measure CYP1A in skin and liver biopsies from live harbour seals and to report in vivo BNF-associated CYP1A induction in a marine mammal. The results demonstrate that microsamples collected using minimally-invasive techniques can provide toxicologically-relevant information form marine mammals.     

The conservation of seals in Irish waters: How research informs policy

Ireland's waters represent a valuable resource encompassing productive fishing grounds and important oil and gas reserves. As a species rich marine environment, Ireland's waters are also an important habitat for many species of marine mammal. Specifically, grey seals (Halichoerus grypus) and harbour seals (Phoca vitulina) are widespread in Irish coastal waters and at least 24 species of cetacean have been reported at sea. The coastal zone represents critical habitat for many of these species, in particular seals that use both terrestrial and aquatic coastal habitat and are vulnerable to habitat degradation. Increasing exploitation of marine resources has increased their exposure to a range of anthropogenic effects including pollution and habitat loss. As top predators seals interact with commercial fisheries both directly and indirectly for resources and conflicts arise. The relatively recent move towards ‘greener’ resource exploitation such as marine renewable energy does not come without negative consequences for marine mammals including seals, which are afforded protection under national and international conservation legislation, highlighting the difficulties faced by resource managers. The sustainable exploitation of resources whilst conserving biodiversity is a challenging task and effective management implementation depends on appropriate policy informed by reliable scientific data. This paper outlines the conservation needs of seals in Irish waters, reviews our current knowledge of these species in Irish water, provides a critical analysis of the existing legislative framework and describes how research outputs can inform present and future policy decisions.     

Molecular cloning of superoxide dismutase (Cu/Zn-SOD) from aquatic molluscs

The potential of the first line of the active oxygen-scavenging system, partial cDNA encoding Cu/Zn superoxide dismutase (SOD) was isolated in three aquatic mollusc species: Ruditapes decussatus (marine clam), Dreissena polymorpha (continental water mussel) and Bathymodiolus azoricus (hydrothermal vent mussel). These SOD cDNA fragments were amplified by PCR with degenerate oligonucleotide primers derived from the amino acid sequence conserved in the Cu/Zn-SOD from several other organisms. A partial cDNA of CuZn-SOD was obtained for R. decussates (510 bp), D. polymorpha (510 bp) and B. azoricus (195 bp). The deduced amino acid sequence showed high similarity among the three mollusc species (57-63%) and among other species (50-65%). The residues involved in coordinating copper (His-47, 49, 64, 121) and zinc (His-64, 72, 81 and Asp-84) were well conserved among the three Cu/Zn-SOD sequences.     

Perceptions and costs of seal impacts on Atlantic salmon fisheries in the Moray Firth,Scotland: Implications for the adaptive co-management of seal-fishery conflict

The Moray Firth Seal Management Plan (MFSMP) was introduced in Scotland in 2005 as a pilot for resolving conflict between Atlantic salmon fisheries and conservation imperatives for protected harbour and grey seals. This adaptive co-management model is now being applied nationally through the Marine (Scotland) Act (2010). However, no information exists on salmon fishery stakeholders’ perceptions of seal predation impacts and related costs, which could influence the success of the MFSMP and other similar initiatives. In 2006 a questionnaire survey of the 95 salmon rod fisheries in seven major Moray Firth rivers was undertaken, and all 20 active salmon netting stations in the Firth. Forty-five fishery owners, 39 ghillies, 120 anglers and 11 netsmen (representing 17 netting stations) responded. The majority (81%) believed that seals had a significant or moderate impact on stocks and catches, 77% believed that all seals were responsible and 47% supported seal culling. Seals were sighted by 38% of rod fisheries, and 18% lost angler days from seal interference. Overall, 0.2% of total reported angler days were lost annually. The estimated direct cost of seal interference for responding rod fisheries was £14,960 annum⁻¹, and losses of catches and damage to nets was £16,500 annum⁻¹ for responding netsmen. Stakeholders’ perceptions were largely inconsistent with their low direct costs and the aims of the MFSMP. Possible reasons for this are discussed, and implications for the governance of future adaptive co-management initiatives for seal-fishery conflict.     

Distribution of cytochrome P4501A (CYP1A) in the tissues of Baltic ringed and grey seals

Information about the expression of CYP1A in wildlife species is essential for understanding the impact of organochlorine exposure on the health status of an exposed population. Therefore, we aimed at characterising a putative CYP1A enzyme expression in both hepatic and extrahepatic tissues of ringed and grey seals from the Baltic Sea and from less polluted waters. The cellular localisation of CYP1A was identified using a monoclonal antibody against scup P4501A1 (MAb 1-12-3). Immunohistochemical staining showed the highest level of CYP1A expression in liver hepatocytes, and the second highest level in the endothelial cells of capillaries and larger blood vessels in the liver and other organs. The most frequent and strongest staining was found in Baltic ringed seals. Although CYP1A-positive staining was observed in only a few tissues in the other seal populations, it was more intense in Baltic grey seals than in Canadian grey seals. The CYP1A enzyme activity, expressed as ethoxyresorufin O-deethylation (EROD), followed a similar tissue distribution and geographical pattern as the immunohistochemistry with clearly elevated EROD activities in most tissues of both Baltic seal populations. Immunochemical characterisation by immunoblotting confirmed the presence and elevation pattern of a putative CYP1A protein in ringed and grey seals, supporting our findings using other methods. The evenly distributed elevation of CYP1A expression among most of the tissues examined indicates that Baltic seals are exposed to CYP1A inducing agents affecting the whole body. This may result in an increased or decreased toxic potential of foreign substances, which may ultimately determine the biological effects of the contaminants.