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.     

The feasibility of assessing the diets of minke whale (<Emphasis Type="Italic">Balaenoptera acutorostrata</Emphasis>) in the East Sea through fatty acid composition in blubber and stable isotopic ratio of skin

To track the diet of minke whale (Balaenoptera acutorostrata) in the East Sea (Japan Sea), a conjoint analysis of fatty acids and C and N stable isotopes was performed on blubber and skin from the whale and its potential prey. The total lipid content in the blubber of minke whales ranged from 37.9% to 82.7% of wet mass (mean ± SD, 63.1 ± 17.2%), with triacylglycerols being the dominant lipids (96.9%–99.2% of total lipids). The lipid and fatty acid (FA) contents were systematically stratified throughout the depth of the blubber layers; contents of the dominant monounsaturated FAs (MUFAs), including 18:1ω9 and 16:1ω7, increased from the innermost layer to the outermost layer, whereas contents of saturated FAs (SFAs) and polyunsaturated FAs (PUFAs) were higher in the innermost layer than in the outermost layer. This stratification is related to the different physiological roles of the blubber layers; e.g., thermoregulation, streaming, and buoyancy. A comparison of the FA compositions of the innermost layer of minke whales with those of potential prey indicates that FA compositions in the whales are similar to those of Pacific herring. In addition, stable isotope ratios (δ¹³C and δ¹⁵N) suggest that minke whale and Pacific herring have the same or similar diets. Therefore, the diets of minke whale from the East Sea (Japan Sea) could be inferred from information on the diet of the Pacific herring, although FA compositions and stable isotope ratios for Pacific herring would not exactly reflect the whale’s diet. Although the very limited number of samples was used in this study, our preliminary findings are very promising to help understand the feeding ecology of minke whales in the East Sea (Japan Sea).     

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].     

Cloning a new cytochrome P450 isoform (CYP356A1) from oyster Crassostrea gigas

We have cloned the full-length cDNA of the first member of a new cytochrome P450 (CYP) family from the Pacific oyster Crassostrea gigas. This new CYP gene was obtained based on an initial 331bp fragment previously identified among the list of the differentially expressed genes in oysters exposed to untreated domestic sewage. The full-length CYP has an open reading frame of 1500bp and based on its deduced amino acid sequence was classified as a member of a new subfamily, CYP356A1. A phylogenetic analysis showed that CYP356A1 is closely related to members of the CYP17 and CYP1 subfamilies. Semi-quantitative RT-PCR was performed to analyze the CYP356A1 expression in different tissues of the oyster (digestive gland, gill, mantle and adductor muscle). Results showed slightly higher CYP356A1 expression in digestive gland and mantle, than the other tissues, indicating a possible role of the CYP356A1 in xenobiotic biotransformation and/or steroid metabolism.     

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).     

小鳁鲸的一些生物学测量数据

2004年6月山东省威海石岛的渔民偶然误捕了一头须鲸,经作者鉴定,此标本系小鳁鲸(Balaenoptera acutorostrata)。主要鉴别特征:头背面的嵴明显;左右鳍肢各有一白色的横斑;有282对白色的鲸须板。同时还对其进行了较详细的观察和测量,值得一提的是此鲸存有异常现象:有一假脐,乳沟位于生殖裂后端而非生殖裂的两旁。     

Expressed sequence tag analysis and cloning of trehalose-6-phosphate synthase gene from marine alga Laminaria japonica (Phaeophyta)

A high quality cDNA library was constructed from the brown alga Laminaria japonica,with the titer of 1.2×10 5 pfu/ml.The average insert size of the cDNA library is about 1.6 kb.From the cDNA library,591 cDNA clones were randomly selected and sequenced.As a result,574 EST(expressed sequence tag) sequences were generated.All of the 574 ESTs were submitted to the dbEST database section of GenBank with the accession numbers from CX942625 to CX943198.The cDNA library was screened with a α-32 p labeled 453 bp T P S gene probe,which is a partial sequence yielded from Porphyra yezoensis.Four positive cDNA clones were screened and the sequencing data showed that these four cDNA clones covered majority of L.japonica TPS cDNA sequence.After PCR amplification,sequencing and assembling,the entire ORF(open reading frame) sequence of the T P S gene was obtained,which was named LjTPS.LjTPS encodes a protein containing 908 amino acids with a calculated molecular mass of 101 674 Daltons.The LjTPS gene was successfully expressed in E.coli and rice.The LjTPS gene has potential application both in plant breeding to stress tolerance and in deciphering the T P S gene function and mechanism to stress tolerance.     

Characteristics of the hepatic microsomal cytochrome P-450 system of the minke whale (Balaenoptera acutorostrata)

The whales are an interesting group in several aspects of science, including evolutionary biology, marine ecology and toxicology. Some of the whale species, including the minke whale, graze at the top of the food chains, where they become susceptible to the accumulation of marine pollutants ingested by organisms at lower levels. The northern stock of the minke whale grazes in Arctic waters in the summer, an area of increasing interest in the development of new oil fields. Studies on the metabolism, disposition and effects of xenobiotics in marine mammals are few. The only reports in the open literature so far have dealt with these questions in seals.^1–3 Although the marine mammals comprise a particularly interesting group in marine ecology and toxicology, no data have to our knowledge been presented on the xenobiotic metabolising enzyme systems of the whales. In this study, components of the microsomal cytochrome P-450 system were measured in liver samples from several females, one male, and two foetal minke whales, caught in the Norwegian whaling season of 1983. Because of the limited number of samples studied we have treated the samples individually and not performed any statistical analysis of the data. The trends discussed below must therefore be considered with this background.     

Gene structure of the novel cytochrome P4501D1 genes in stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes)

The cytochrome P450 1 (CYP1) family has expanded with the addition of the CYP1B and CYP1C subfamilies. We recently identified a new CYP1 subfamily in zebrafish, CYP1D, with a single gene, CYP1D1. Here we examined sequences found in other fish genomes, i.e., stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes), for similarities among fish CYP1D1 genes. The full-length deduced amino acid sequences for CYP1D1 in these two species averaged about 43% identity to the CYP1As, but nearly 50% when sequence alignment ambiguities were masked. CYP1D1 has seven exons, similar in size and position to the exons in CYP1D1 and CYP1A in zebrafish. However, the intronic distances were substantially smaller in the medaka and stickleback. There also were differing numbers of putative xenobiotic response elements in the CYP1D1 of the various species. Whether the stickleback or medaka genes are inducible by aryl hydrocarbon receptor (AHR) agonists is yet to be determined.     

A report on the Norwegian minke whale hunt

The Norwegian minke whale hunt was resumed in 1993, despite considerable international opposition. This paper investigates economic aspects of the 1993–1996 hunts. It is shown that the hunts were profitable for the participating vessels. Adverse international effects due to the resumed hunt are discussed, as is the future of commercial whale hunting in Norway.     

刺参肌肉组织cDNA文库的构建

用RNA提取试剂--TRIZOL Reagent提取刺参(Apostichopus japonicus)肌肉组织总RNA,用SMART cDNA Library Construction Kit构建cDNA文库.经测定原始文库滴度达到 3.2×10~6,扩增后文库滴度达到 5.1×10~9,重组率达到96.7%,从扩增文库随机挑取12 个克隆进行PCR 扩增鉴定,结果显示,插入片段大小为0.5～2.5 kb.通过各项指标验证成功构建了刺参肌肉组织cDNA 文库.     

Gene discovery in the tropical scallop Nodipecten nodosus: Construction and sequencing of a normalized cDNA library

We report the construction and characterization of a normalized cDNA library from the digestive gland of the marine bivalve Nodipecten nodosus, a commercially valuable tropical scallop. A total of 288 clones were sequenced, and 250 unique sequences were obtained. The cDNA library showed a small sequence redundancy (2.3%) and high numbers of recombinant (99.9%) and independent clones (2.0 × 10⁶ cfu), indicating that the cDNA library generated in this study is a profitable resource for efficient gene discovery for N. nodosus. EST functional annotation by Gene Ontology term assignment revealed the identification of sequences potentially involved in aquaculture and ecotoxicology relevant processes such as apoptosis, growth, lipid metabolism, reproduction, development, response to stress and immunity.     

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.     

Modulation of trout 7-ethoxyresorufin-O-deethylase (EROD) activity by estradiol and octylphenol.

Estrogens appear to have a modulating effect on the expression of cytochrome P4501A (CYP1A) in fish. A number of in vivo studies have demonstrated that hepatic CYP1A expression in females decrease during sexual maturation when plasma levels of 17 beta-estradiol (E2) increase, or in cases when the fish in injected with E2. Since a number of environmental contaminants have weak estrogen-like activities, the question arises if these compounds are able to modulate CYP1A expression as well. In the present study, we used in vitro monolayer cultures of rainbow trout, Oncorhynchus mykiss, liver cells to compare concentration-dependent (10(-9) to 10(-5) M) effects of the natural steroid E2 and the non-steroidal xenoestrogen 4-tert-octylphenol (OP) on CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity. The concentration dependency of the estrogenic activity of the two test compounds was assessed by determination of hepatocellular vitellogenin (Vg) release into the culture medium. Exposure of hepatocytes to E2 concentrations of 10(-8) M and higher led to a significant inhibition of basal cellular EROD activity. On the contrary, exposure to OP did not result in an inhibition of EROD activity, even at OP concentrations (10(-6) M, 10(-5) M) which were associated with a significant induction of Vg synthesis.     

Using trace elements in skin to discriminate the populations of minke whales in southern hemisphere

Concentrations of 12 trace elements (V, Cr, Mn, Cu, Zn, Se, Rb, Sr, Cd, Cs, Ba, and Hg) were determined in liver and skin tissues of minke whales from various regions within the Antarctic Ocean. Cd concentrations in livers of southern minke whale were apparently higher than those in cetaceans from other regions, while Hg concentrations were lower. There were significant positive correlations between body length and concentrations of Cd and Hg in the liver. The concentrations of all trace elements in the skin were lower than those in other cetaceans reported previously. Significant positive correlations between liver and skin were found for Cr, Mn, Cu, Zn, Rb, Cd, and Cs, implying that the concentrations of these trace elements in the skin reflect those of internal organs. Large interannual variation of the accumulation pattern of trace elements in the skin was observed for the southern minke whales from Area V. There were significant differences in the skin element concentrations among Areas III, IV, and V, especially for males. Also, discriminant analysis between geographically two different groups collected during 1995/1996 austral summer season, based on the concentrations of trace elements in the skin, allowed for a correct classification of 90% of these minke whales. These results suggest that measurement of trace elements in skin samples could provide valuable information on the status of contamination and possible geographic differences in the accumulation levels in southern minke whales.     

Biomarker induction in tropical fish species on the Northwest Shelf of Australia by produced formation water

Normal operation of oil well platforms results in the discharge of produced formation water (PFW). The expression of CYP1A, CYP2M1- and 2K1-like proteins was examined for use as possible biomarkers of PFW exposure. A pilot study on the Northwest Shelf of Australia had indicated that PFW contamination possibly contributes to induction of CYP1A-like proteins in Gold-Spotted Trevally (Carangoides fulvoguttatus). The pilot study samples were re-examined for CYP1A, and, in addition, CYP2K1/2M1-like proteins. In a subsequent caged fish study in the same location a second species, Stripey seaperch (Lutjanus carponotatus), caught at a clean site, were distributed to three caging sites in a PFW gradient from the Harriet A production platform: A (near-field), B (far-field) and C (a non-impacted reference site). Fish were sampled at time (T) T = 0, T = 3 and T = 10 days. Significant increases of CYP1A, one CYP2K1- and two CYP2M1-like proteins were noted at Site A at T = 10d. For another CYP2K1-like protein, a significant increase was observed at Site A only at T = 3d. These results support a previous study indicating that CYP1A protein is sensitive to PFW exposure. Importantly, statistically significant environmental induction of both CYP2M1- and CYP2K1-like proteins in tropical fish due to PFW exposure had not previously been described and induction of enzymes in the CYP2 family suggest new biomarkers for PFW. In addition, the novel response of one CYP2K-like protein requires further verification, but offers promise for improved monitoring of sub-lethal responses in marine organisms.