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.     

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).     

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.     

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].     

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.     

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.     

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。     

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。     

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.     

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.     

黄斑篮子鱼去毒相关基因的克隆与肝脏组成型表达分析

从基因水平探讨海洋鱼类对海洋藻毒素的去毒分子机理。采用RT-PCR法成功克隆了黄斑篮子鱼Siganus oramin肝脏I时相代谢酶细胞色素P450 1A(CYP1A)、II时相代谢酶alpha型谷胱甘肽S-转移酶(GSTA)和rho型谷胱甘肽S-转移酶(GSTR)、热休克蛋白70 (HSP70)、alpha 1型钠钾ATP酶(ATP1A1)及β-肌动蛋白(beta-actin, ACT)基因cDNA核心序列,序列分别长879 bp、582 bp、588 bp、660 bp、749 bp和554 bp。序列同源性分析发现,属鲈形目的黄斑篮子鱼CYP1A、GSTA和GSTR与同属鲈形目的牙鲆Paralichthys olivaceus、欧洲鲽Pleuronectes platessa、真鲷Pagrus major、鲤形目的斑马鱼Brachydanio rerio 相应氨基酸序列同源性较高,CYP1A和GSTA与非洲爪蟾(两栖类)、鸡(鸟类)、小鼠、大鼠和人(哺乳类)相应氨基酸序列同源性低,这可能与鱼类I、II时相去毒酶基因承担水环境毒素去毒代谢的特殊功能有关;而HSP70、ATP1A和β-肌动蛋白在鱼类、两栖类、鸟类、哺乳类中均有较高的同源性,这可能与这些基因在机体中承担的最基本的生命功能相关。应用半定量RT-PCR的方法,以β-肌动蛋白作为外参照,在指数期增长范围内分别得到了CYP1A、GSTA、GSTR、HSP70和ATP1A1 mRNA与β-肌动蛋白mRNA (%)的比值,确定黄斑篮子鱼肝脏去毒相关基因的组成型表达水平。其中,黄斑篮子鱼肝脏CYP1A、GSTA和GSTR基因组成型表达相对较高,HSP70和ATP1A1基因组成型表达相对较低,这可能与不同基因在黄斑篮子鱼海洋藻毒素去毒分子机理中承担的作用相关,为海洋藻毒素在海洋鱼类中的积聚及代谢去毒分子机制的研究提供了相关数据。     

The effects of seal-swim activities on the New Zealand fur seal (Arctophoca australis forsteri) in the Bay of Plenty,New Zealand,and recommendations for a sustainable tourism industry

Wildlife tourism (including pinniped tourism) offers people the opportunity to see wildlife in their natural environment. It can provide positive outcomes for the animals, through improved resources for conservation, or negative outcomes, such as inducing the animals to move away. This study assessed the impacts and sustainability of a novel but growing tourism industry, swimming with seals, based on interactions with New Zealand fur seals (Arctophoca australis forsteri) in the Bay of Plenty, New Zealand, between December 2011 and March 2012. The behaviour of all seals in the water (interaction, neutral, and avoidance) was monitored at 1 min intervals, during 16 seal-swim events. Seals mostly ignored the swimmers (54% of records), some interacted with swimmers (41%); seals rarely avoided the swimmers (5%). Interactions peaked in frequency at 6 min into the swims, then declined. They occurred most frequently during December, corresponding with the pupping period when juvenile seals—the age class most likely to interact—are excluded from breeding areas and so spend much of their time in the water. Compliance of tour operators to regulations was also monitored during seal-swim activities and the industry was found to be highly compliant. The results suggest the activities monitored had minimal impact on seals in the water, and are likely to be sustainable in relation to seal conservation. Tourism can be site and time specific, and it is recommended that approaches such as those trialled here be adopted to monitor other wildlife tourism activities to ensure their sustainability. Further research needs to examine potential impacts of the tours on seals ashore.     

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.     

Apparent lack of CYP1A response to high PCB body burdens in fish from a chronically contaminated PCB site

Chronic exposure to organic contaminants such as polychlorinated biphenyls (PCBs) can lead to the development of resistance to these chemicals, a condition associated with reduced response of CYP1A1, a pollutant-inducible biomarker. We measured CYP1A activity (ethoxyresorufin o-deethylase, EROD) and PCB concentrations in feral fish from the Town Branch/Mud River system (Logan County, KY), a stream historically contaminated with PCBs and partially remediated. As a first step in evaluating the possible development of resistant populations in this system, we measured CYP1A expression and PCB body burdens in resident fish from sites we previously characterized as containing biologically significant levels of CYP1A inducing compounds. Mean PCB concentrations in edible flesh ranged from 75.2 to 16.7 microg/g in fish collected from Town Branch remediated sites and were relatively low (1.23 microg/g) in Town Branch reference site fish. However, hepatic CYP1A activity was similar among individuals of most species collected from reference and contaminated/remediated sites. The absence of elevated CYP1A levels in resident fish species despite the presence of significant PCB body burdens may indicate these fish have developed reduced sensitivity to CYP1A induction, a condition associated with acquired resistance to toxicants.     

The Japanese medaka (Oryzias latipes) model: applicability for investigating the immunosuppressive effects of the aquatic pollutant benzo[a]pyrene (BaP)

Despite the fact that BaP is a carcinogen, mammalian immunosuppressant, and ubiquitous aquatic pollutant, knowledge regarding the effects of BaP on the immune system of fish is still lacking. To begin to fill this gap, studies were conducted in medaka to examine the effects and mechanisms by which BaP exposure might alter host immunocompetence. Fish, exposed by IP injection of BaP (2-600 microg/g BW), were examined after 48 h for effects upon immune function and CYP1A expression/activity. Benzo[a]pyrene, at a concentration below that which increased levels of CYPIA expression/activity (2 microg BaP/g BW) suppressed lymphocyte proliferation. Concentrations of BaP at 20 and 200 microg/g BW. suppressed antibody-forming cell (AFC) numbers, superoxide production, and host resistance against bacteria. In contrast, exposure to the low affinity aryl hydrocarbon receptor (AhR) agonist, benzo[e]pyrene (BeP), neither induced CYP1A expression nor altered immune function. Given the lack of immunosuppressive effects produced by BeP, and the fact that exposure to the AhR antagonist (and CYP1A inhibitor) alpha-naphthoflavone (ANF) ameliorated the suppressive effects of BaP upon AFC numbers, the AhR pathway (including CYP1A-mediated production of reactive BaP metabolites) appears important in mediating BaP-induced immunotoxicity in fish, as in mammals. In the past, the medaka has proven a successful model for assessing carcinogenic agents. These studies have demonstrated its utility for also determining the immunosuppressive effects of an important aquatic contaminant.     

Searching for novel CYP members using cDNA library from a minke whale liver

The contaminant-induced cytochrome P450 (CYP) members in minke whale (Balaenoptera acutorostrata) can be potential biomarkers of the contaminant exposure and toxic effects. In this study, we constructed a cDNA library from the liver of minke whale from the North Pacific, and further screened a total of 6930 clones randomly selected in the library for the isolation of cDNA clones encoding novel members of CYP superfamily. The screening revealed the isolation of six novel CYP cDNA clones that are classified into CYP1A, CYP2C, CYP2E, CYP3A, CYP4, and CYP4A subfamilies. The BLAST homology search using the partial cDNA fragments of four CYP subfamilies (CYP1A, CYP2C, CYP2E and CYP4A) demonstrated that the minke whale CYPs were most closely related to pig CYPs (81-91%). Identification of multiple CYP genes in marine mammal species such as minke whale will provide new insights into the metabolic or toxicological functions of individual CYP members.     

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.     

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.