cDNA cloning of an aryl hydrocarbon receptor from Baikal seals (Phoca sibirica)

Species differences in sensitivity to related planar halogenated aromatic hydrocarbons (PHAH) add significant uncertainty in assessing the ecological risk to aquatic mammals. To investigate mechanisms of PHAH sensitivity in aquatic mammals, we cloned and sequenced the cDNA of Baikal seal aryl hydrocarbon receptor (AHR), an intracellular protein that initiates PHAH-mediated effects. The Baikal seal AHR cDNA has an open reading frame of 843 amino acid residues with a predicted molecular mass of 94.6 kDa. Comparison of AHR amino acid sequences indicated a high degree of sequence conservation (98%) between Baikal and harbor seals. The high conservation of AHRs between Baikal and harbor seals indicates that these seals express AHR proteins closely related structurally. In our previous report (Kim & Hahn, 2002), the dioxin-binding affinity of the harbor seal AHR was at least as high as that of the AHR from a dioxin-sensitive strain of mice, suggesting that this seal species may be sensitive to PHAH effects. This implies that Baikal seal may also be sensitive to dioxin effects.     

Cytochrome P450 gene diversity and function in marine animals: past, present, and future

The biotransformation of xenobiotics by microsomal cytochromes P450 is known to be pivotal in the effects of some compounds, and thought to be so for many. A knowledge of CYP gene diversity and CYP function and regulation in aquatic species is pursued, expecting that it will disclose mechanisms, allow predictions regarding species differences in susceptibility, and provide markers for exposure to xenobiotics. As well, it is hoped that such knowledge will provide clues to CYP endogenous functions, and to the origin and functional significance of CYP gene diversity. The knowledge of CYP in marine and other aquatic species is expanding rapidly. The diversity of CYP genes in non-mammalian vertebrates may approximate that in mammals. At present, cloning studies have identified members of gene families 1 to 4 have been cloned from one or more fish species. Where known, the gene structures of fish CYP genes are like those of mammalian homologues. Only one CYP1A gene has been identified in most fish species examined. Fish CYP1As, including multiple forms from recent divergence in some genera, have structural and catalytic properties more like CYP1A1, but also have properties that are 1A2-like, consistent with fish CYP1As representing the CYP ancestral to both CYP1A1 and CYP1A2. A number of genes cloned from several species have been classified in the 3A subfamily. Fish CYP3As catalyze steroid 6β-hydroxylase, and have other properties consistent with mammalian 3As. Recently identified CYP4 genes classify to novel subfamilies but apparently are homologues of mammalian CYP4 genes, and may act on similar substrates. The greatest diversity of fish CYP genes is in family 2; there are now six fish CYP2 subfamilies known. Four of these are novel subfamilies, although cladistic analysis suggests distinct relationships to mammalian CYP2 subfamilies. Heterologous expression and characterization of some of these CYP have identified similar functions among genes in different subfamilies. For example, fish CYP2Ns and CYP2Ps are related to mammalian CYP2Js, and CYP2P3 and CYP2J2 have strikingly similar functions as fatty acid epoxygenases and hydroxylases, with nearly identical regio- and enantioselectivity for metabolism of arachidonic acid. In addition to sequence and catalytic similarities, there also are indications that CYP regulation, tissue and cellular localization are similar between fish and mammals. Yet even in cases where orthology is strongly suggested, e.g. CYP1A, there appear to be taxonomic differences in active site structure suggesting potential differences in involvement of CYP1A in toxicity. In contrast to fish, CYP diversity and functions in aquatic invertebrates are poorly known. Investigators have identified novel gene families and subfamilies in crustaceans (CYP2L; CYP45), molluscs (CYP30, CYP10) and sponges (CYP38). CYP4C genes occur in crustaceans, molluscs and echinoderms, and a new subfamily (CYP4Y) in molluscs. The future? There is no doubt that new CYP will continue to be discovered in non-mammalian vertebrates; some (e.g. CYP51) can be predicted confidently. And, there is no doubt that the numbers known in invertebrates will expand greatly. In insects and C. elegans the numbers are very high, and even slime molds have 18 CYP genes. It is virtually certain that CYP genes with unique functions will be discovered. While the knowledge of CYP genes is increasing, knowledge of CYP function and regulation lag well behind. Technical approaches to speed the aquisition of such knowledge are available. The information will be essential to discern the role that CYP play in the disposition and toxicity of xenobiotics, during development as well as in adults. Yet, when such data are in hand, we may have to face the paucity of information on the diversity, function and regulation other enzymes, notably the glutathione S-transferases, glucuronyl transferases and sulfotransferases, in aquatic species. Discerning orthologous relationships among CYP genes, as well as those for phase II enzymes, could highlight gene lineages associated with conserved and endogenous functions. Understanding CYP endogenous functions, as well as their metabolism of xenobiotics, may reveal fully the ways that chemicals cause toxicity. [Support: Sea Grant NA46RG0470-R/P61, EPA R-829890, NIH ES07381].     

Identification of cytochrome P450 1B-like sequences in two teleost fish species (scup, Stenotomus chrysops and plaice, Pleuronectes platessa) and in a cetacean (striped dolphin, Stenella coeruleoalba).

The cytochromes P450 (CYP) constitute a multigene family of enzymes playing a critical role in the oxidation of many endogenous and xenobiotic substrates. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and aryl amines. Three members of the CYP1 family, CYP1A1, CYP1A2, and CYP1B1, have been identified in mammals. We report here on the identification and cloning of cytochrome P4501B-like sequences from two teleost fish species and a marine mammal. Sequences clustering with CYP1B1 in phylogenetic analysis were obtained from liver cDNA of scup (Stenotomus chrysops), genomic DNA of plaice (Pleuronectes platessa), and liver cDNA of striped dolphin (Stenella coeruleoalba).     

Entanglement of pinnipeds at Marion Island

During the period April 1991–March 1996, 10 entangled Antarctic fur seals Arctocephalus gazella, 28 entangled Subantarctic fur seals A. tropicalis and one entangled southern elephant seal Mirounga leonina were observed at Marion Island, Southern Ocean. Entanglement of fur seals was estimated at between 0.01 and 0.15% of the combined population of both species.     

大黄鱼(Pseudosciaena crocea)细胞色素P450 CYP4F7基因的克隆及表达分析

CYP4F7是细胞色素P450超基因家族中CYP4F亚家族中的同工酶之一,而细胞色素P450在通过电子传递参与生物体代谢和转化内源和外源化合物方面起着重要的作用。以本实验室构建的大黄鱼消减杂交cDNA文库中623bp的CYP4F7片段设计引物,以大黄鱼的总RNA为模板,利用RACE-PCR的方法,克隆鉴定出了大黄鱼细胞色素P450 CYP4F7基因。结果表明:基因全长1934bp,5'端非编码区59bp,3'端非编码区264bp,开放阅读框1611bp,编码536个氨基酸。序列分析表明大黄鱼的CYP4F7氨基酸序列与舌齿鲈的相似性为91%。利用RT-PCR方法研究CYP4F7在大黄鱼各组织中的表达特征,结果表明CYP4F7在肝脏、脾脏中表达量最高,在心脏、肾脏、头肾、鳃丝中表达量次之。另外,CYP4F7在注射了细菌脂多糖的大黄鱼各组织中的表达量均低于正常的大黄鱼,认为细菌脂多糖可能是CYP4F7表达的抑制剂,说明CYP4F7可能与鱼类免疫反应有一定相关性。     

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.     

Measurements of tagged pups and a population estimate of New Zealand fur seals on Taumaka,open bay Islands,Westland

In January 1970, 309 pups of the New Zealand fur seal, Arctocephalus forsteri (Lesson, 1828), were tagged on Taumaka Island, one of the Open Bay Islands, Westland, New Zealand (43°52’ S, 168°53’ E). At about 6 weeks old, male pups have longer flippers and are longer and heavier than female pups.

A population estimate of seals on Taumaka Island is based on a count of pups of the year, to which data from related species of fur seals were applied (incidence of pregnancy and age at first pupping from Arctocephalus pusillus, immature mortality from Callorhinus ursinus), and on the assumptions that the sex ratio and sexual mortalities are equal. There were at that time 2,000–3,000 seals, including pups, on the island.     

The use of polyclonal antibodies raised against rat and trout cytochrome P450 CYP1A1 orthologues to monitor environmental induction in the channel catfish (Ictalurus punctatus)

Induction of hepatic cytochrome P450-dependent microsomal mono-oxygenase by xenobiotics is a well-established phenomenon in teleost fish. As in laboratory mammals, fish possess multiple forms of cytochrome P450 that display overlapping substrate specificity. One such isoform, CYP1A1, which has been cloned and sequenced from rainbow trout, has been shown to be orthologous to rat CYP1A1 and, as in mammals, is inducible up to several hundred-fold by planar aromatic hydrocarbons, PCBs and dioxins. It has been suggested that induction of CYP1A1 orthologues might provide a sensitive biomonitor for environmental pollution by mixtures of such compounds. In the current study, polyclonal antibodies directed against CYP1A1 purified from rat and trout liver were used to monitor induction of the CYP1A1 orthologue in hepatic microsomes from the fresh water species, the channel catfish (Ictalurus punctatus). Catfish from a local fish farm were induced in the laboratory by three daily injections of 50 mg/kg of the PCB mixture Aroclor 1254 and compared with fish taken from a site in central Arkansas—the Bayou Meto, known to be polluted with dioxin. Hepatic microsomal activities towards ethoxyresorufin (EROD) and pentoxyresorufin (PROD) were measured and Western blot analysis carried out with the two antibodies. EROD was elevated in both the Aroclor-treated fish and in the Bayou Meto fish compared with untreated fish farm controls; smaller but significant increases were observed in PROD. Spearman's rank correlations of 0·74 and 0·89 were observed between EROD and immunoquantified cross-reactivity towards the rat CYP1A1 and trout CYP1A1 antibodies.     

Population trends of New Zealand fur seals in the Rakiura region based on long-term population surveys and traditional ecological knowledge

We estimated population abundance of New Zealand fur seal (Arctocephalus forsteri) pups on Bench Island off Stewart Island, New Zealand seven times between 1996 and 2012. Overall, there was a 29% increase in pup abundance from 1996 to 2012 at the Main Beach colony, corresponding to a mean annual growth rate of 1.6% and a doubling time of approximately 40 years. At the Sprat Point colony, there was an overall increase of 29% between 2003 and 2012 corresponding to a mean annual growth rate of 2.9% and a doubling time of approximately 25 years. The area occupied by both colonies has also increased. In 2006, we surveyed East Beach and counted a total of 201 pups. We obtained traditional ecological knowledge of fur seal distribution and breeding status from local Māori for 46 locations around Stewart Island, 36 of which have not been surveyed since Wilson in 1971–1974; this supports an expansion of fur seal presence and breeding areas in the region in the last 41 years.     

Summer survey of fur seals at Prince Edward Island,southern Indian Ocean

The onshore distributions and the abundances of Antarctic fur seals Arctocephalus gazella and Subantarctic fur seals A. tropicalis were determined at Prince Edward Island during 16-20 December 2008. This repeats a survey conducted in December 2001 and extends the area surveyed to include the entire south-west coast of Prince Edward Island. Of the two colonies of Antarctic fur seals, the colony among Subantarctic fur seals north of Boggel Beach remained small, with increased numbers of Subantarctic fur seals and putative hybrids. The other Antarctic fur seal breeding colony at Penguin Beach remained free of Subantarctic fur seals and had expanded at a mean intrinsic rate of natural increase of 11.4% per year from 2001. With an estimated 810 pups, the Antarctic fur seal is still in the rapid recolonisation phase of population growth. The distribution of the more widespread and abundant Subantarctic fur seals also had increased, with several new breeding colonies along the east coast and one at Kent Crater on the west coast. The annual pup production was conservatively estimated at 14 130 pups. The mean intrinsic rate of natural increase has declined to ?0.3% per year over the last seven years, compared to the 9.3% per year between 1987/1988 and 2001/2002, and the population is in the mature phase of population growth.     

Indications of P450 monooxygenase activities in beluga (Delphinapterus leucas) and narwhal (Monodon monoceros) from patterns of PCB, PCDD and PCDF accumulation

Selective accumulation of PCDDs, PCDFs and PCB congeners was investigated in beluga (Delphinapterus leucas), narwhal (Monodon monoceros) and several species at the same trophic level in two areas of Canada. There was a much higher relative abundance of meta-para-unsubstituted PCBs in the cetaceans compared with other species. This suggests that activity of cytochrome P450 monooxygenase enzymes causing CYP2B-type metabolism was relatively low in beluga and narwhal. Lower relative levels of 2,3,7,8-TCDD occurred in beluga from both areas, and selective reduction of toxic non-ortho PCBs occurred in the highly PCB-contaminated St Lawrence beluga. These compounds are potent inducers of cytochrome P450 CYP 1A-type enzymes, suggesting that such an enzyme with the capability of metabolizing TCDD-like substrates is present in beluga and narwhal. SIMCA principal components analysis of PCB congener patterns showed that the two cetaceans were in a statistically significant different class than the other species. Variables (congeners) with the greatest separation power between classes were mainly those defined by the selective metabolism rules.     

Subsistence fisheries in South Africa: a Preface

A juvenile Subantarctic fur seal Arctocephalus tropicalis was recorded on the Indian Ocean island of Rodrigues and two were seen on Mauritius. These records are at least 2 500 km from the closest breeding colony (Amsterdam Island) and are the most easterly by some 1 800 km of all previous records of vagrants of this species.     

Diet of the Cape fur seal Arctocephalus pusillus pusillus at the Robberg Peninsula,Plettenberg Bay,and implications for local fisheries

Cape fur seals Arctocephalus pusillus pusillus were harvested to extirpation on the Robberg Peninsula, Plettenberg Bay, on the south-east coast of South Africa, between the 17th and early 20th centuries. Seals returned to Robberg in small numbers during the early 1990s and their numbers subsequently increased. We studied the diet of this increasing population using faecal (scat) sampling to determine: the species composition and size of prey in the diet of Cape fur seals at Robberg; to explore temporal variation in the diet; and to investigate the potential for competition between seals and the fisheries around Plettenberg Bay. Of the 445 scats collected, 90% contained hard prey remains and 15 teleost prey species were represented in the 3 127 otoliths that could be identified. The seals’ most important prey species in terms of numerical abundance, frequency of occurrence and mass in the diet, were anchovy Engraulis encrasicolus, sardine Sardinops sagax, horse mackerel Trachurus capensis, sand tongue-fish Cynoglossus capensis and shallow-water hake Merluccius capensis (in decreasing order of importance for numerical abundance). The proportion of anchovy in the diet increased during the study period (2003–2008), whereas the proportion of sardine decreased. The estimated average annual consumption of sardine by seals was higher than the average annual catch made by purse-seine fisheries in this area, suggesting resource competition between seals and purse-seiners, especially in the light of continuing growth of seal numbers in the area. However, direct competition between seals and linefisheries appeared to be minimal. Scat sampling of Cape fur seals holds potential to serve a useful and cost effective indicator of temporal changes in sardine abundance.     

Population Numbers of Fur Seals at Prince Edward Island,Southern Ocean

During the period 17–22 December 2001, the onshore distribution and the abundance of Antarctic fur seals Arctocephalus gazella and Subantarctic fur seals A. tropicalis were determined for Prince Edward Island. Two breeding colonies of Antarctic fur seals were located on the south-east coast of the island; the first a mixed (with Subantarctic fur seals) breeding colony with an estimated 24 pups on a vegetated promontory on the northern section of Boggel Beach, and the second, a presumably pure Antarctic fur seal breeding colony with an estimated 380 pups, at Penguin Beach. At a mean intrinsic rate of natural increase of 16.2% per year, Antarctic fur seals appear to be in the rapid recolonization phase of population growth. Breeding colonies of Subantarctic fur seals, largely found on the entire east coast, produced an estimated 15 000 pups, and the population had maintained a mean intrinsic rate of natural increase of some 9.5% per year since 1987/88.     

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.     

Variation in levels of cytochrome P4501A, 2B, 2E, 3A and 4A-immunopositive proteins in digestive gland of indigenous and transplanted mussel Mytilus galloprovincialis in Venice Lagoon, Italy

Mytilus galloprovincialis digestive gland microsomes were prepared from (i) indigenous populations sampled from a clean reference (Lido) and an urban-contaminated site (Salute) and (ii) mussels transplanted for up to 3 weeks from Lido to an industrial-contaminated site (CVE) in Venice Lagoon, Italy. Western blot analysis was performed using antibodies to five mammalian or fish CYP forms (1A, 2B, 2E, 3A, 4A). Simultaneously run M. edulis digestive gland partially purified CYP aided identification of immunopositive bands. Levels of CYP1A, CYP2E, and CYP4A-immunopositive proteins were 50 to 300% higher in indigenous M. galloprovincialis from Salute compared to Lido (p < 0.05). Three weeks after transplantation to CVE, levels of only the CYP1A-immunopositive protein were determined to be higher (63%) than levels for Lido (p < 0.05), indicating that anti-CYP1A shows greater specificity for a contaminant-inducible CYP form than the other antibodies.     

Isolation of CYP2L2 and two other cytochrome P450 sequences from a spiny lobster, Panulirus argus, hepatopancreas cDNA library

Cytochrome P450 monooxygenases constitute an enzyme superfamily, with at least 74 known families. Members of CYP families 1–4 are important in the phase 1 metabolism of lipophilic xenobiotics, such as those found in contaminated marine environments. Previous studies (James et al. Arch. Biochem. Biophys. (1996) 329, 31–38) showed that a major form of P450 in spiny lobster, Panulirus argus, hepatopancreas was CYP2L1, a new sub-family, and that there was evidence for other P450 forms in hepatopancreas. We now report the sequence of a second member of this subfamily, named CYP2L2, present in P. Argus hepatopancreas. The deduced amino acid sequences of CYP2L1 and CYP2L2 share 54.7% sequence identity and an additional 13.6% of the sequences show conservative substitutions. Analysis of the sequences of CYP2L1, CYP2L2 and other representative CYP2 family members (from rat and mouse sub-families 2A, 2B, 2D and 2E) showed that the crustacean sequences clustered together. In addition to CYP2L2, cDNA clones of 66 to 117 base pairs from the 5′ coding region of two more P450 isoforms were isolated from the spiny lobster cDNA library. The deduced amino acid sequence of one of these additional cDNA clones was identical to the first 22 amino acids of the N-terminal sequence of a P450 protein previously isolated from hepatopancreas microsomes. These studies confirm earlier biochemical evidence that the hepatopancreas contains multiple forms of cytochrome P450.     

Comparison of cytochrome P450 in three butterflyfish species: Ecological implications of elevated CYP2B and CYP3A in Chaetodon capistratus

The relationship between cytochrome P450 and feeding on terpenoid-rich gorgonian corals was investigated in a species of tropical butterflyfish and compared with two other sympatric congeners that do not feed on gorgonians. Fish were collected from non-polluted waters in Belize and the levels of two cytochrome P450 isozymes (CYP2B and CYP3A) were immunoquantitated in addition to quantification of total P450. Chaetodon capistratus regularly feeds on gorgonian corals and has higher levels of total hepatic microsomal cytochrome P450 than C. ocellatus or C. striatus. The content of hepatic P450 (0.588–0.794 nmol mg⁻¹) in C. capistratus is among the highest ever reported in teleosts from non-polluted waters and is significantly greater than detected in C. ocellatus or C. striatus. Chaetodon capistratus also had a larger hepatic index (g liver per g fish) and more microsomal protein (mg protein per g liver), factors that translate into 3.3- to 8-fold more total P450 per g fish. Sexual differences in total P450 were observed between male and female C. capistratus, but not among the other species. The contents of proteins detected by immunoassay with polyclonal anti-scup P450B (CYP2B) and anti-human P4503A (CYP3A) were 2- to 10-fold and 2- to 20-fold greater, respectively, in C. capistratus than in the congeneric species. CYP2 and CYP3 gene families in mammals are thought to have evolved partially in response to dietary allelochemicals. These results suggest that these P450 isozymes may also be important in marine teleosts that feed on terpenoid-rich prey.