Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

长牡蛎新型模式识别受体CgLRRC69的克隆和功能鉴定

在双壳类软体动物中, 血淋巴细胞介导的吞噬作用是清除入侵微生物的主要方式。本文在长牡蛎中鉴定了一个包含富含亮氨酸重复序列(Leucine-rich repeat, LRR)结构域的新型基因, 命名为CgLRRC69。对该基因的组织分布分析表明, CgLRRC69 mRNA在血淋巴细胞、鳃、肌肉、外套膜、心脏、消化腺和性腺中广泛表达。副溶血弧菌感染可以显著地刺激CgLRRC69在血淋巴细胞中表达, 并且在感染后6h达到峰值。同时, 酶联免疫吸附实验发现CgLRRC69可以特异性结合脂多糖(lipopolysaccharide, LPS), 表明它可能在免疫防御中有功能。吞噬实验结果显示, CgLRRC69重组蛋白可以显著地提高血淋巴细胞的吞噬能力; RNAi干扰CgLRRC69在牡蛎体内的表达, 显著降低了血淋巴细胞对细菌的清除能力。因此, 这些结果揭示了CgLRRC69作为一种新型模式识别受体, 可以特异性识别革兰氏阴性菌的主要成分LPS, 通过调理作用有效地清除细菌。     

Cloning and sequencing of cytochrome P450 2X1 from channel catfish (Ictalurus punctatus)

Previous purification and immunochemical studies in livers of channel catfish indicated the presence of at least four cytochrome P450 (CYP) 2-like isoforms. Sequencing of the first 18 amino acids of one purified form indicated a CYP2 isoform. From this N-terminal sequence and other published CYP2 sequences from fish, primers were designed and a full-length CYP cDNA was identified from reverse-transcribed catfish liver mRNA. 5' and 3' RACE was used to obtain an open reading frame of 1470 bp encoding a 490 amino acid protein (approximately 57 kD). CYP2X1 was most identical to Fundulus heteroclitus CYP2P2 (41%); CYP2N2 (40%): and CYP2N1 (39%).     

Catalytic function of avian cytochrome P450 1A4 and 1A5 (CYP1A4 and CYP1A5) enzymes heterologously expressed using in vitro yeast system

The present study clarifies the enzymatic properties of two avian cytochrome P4501A (CYP1A) paralogs, CYP1A4 and 1A5, using a yeast-based vector system. Recombinant CYP1A4 and 1A5 proteins from common cormorant (Phalacrocorax carbo) were expressed in yeast cells, and showed typical reduced CO-difference spectra with a peak at 446 nm. Kinetic analysis of O-dealkylase of methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin catalyzed by the CYP1A enzymes revealed that Vmax value for ethoxyresorufin-O-deethylase (EROD) activity was much higher than that for the other three O-dealkylase activities for both isozymes. Interestingly, remarkable substrate specificity of the CYP1As was observed for O-dealkylation of benzyloxyresorufin and methoxyresorufin; CYP1A4 was highly specific for catalyzing benzyloxyresorufin-O-debenzylase activity, whereas CYP1A5 was more efficient in catalyzing methoxyresorufin-O-demethylase activity. The present study also measured CYP1A-dependent EROD activity in the presence of 2,3,7,8-tetrachlorodibenzofuran (TCDF) to evaluate the ability of this dioxin-like congener to inhibit the EROD activity. One hundred nanomolar TCDF noncompetitively inhibited CYP1A5-dependent EROD activity, although no inhibitory effect was detected for CYP1A4-dependent EROD activity. These results indicate that the avian CYP1A paralogs have different affinities for substrate and inhibitor, thus suggesting their distinct physiological and toxicological roles.     

The cytochrome P450 1A gene (CYP1A) from European flounder (Platichthys flesus), analysis of regulatory regions and development of a dual luciferase reporter gene system.

Concensus primers designed to CYP1A-conserved regions were used to amplify a 1.3 kb probe from flounder genomic DNA via polymerase chain reaction (PCR). A 14-kb clone was isolated from a flounder genomic library constructed in lambda FIXII. Of this clone, 8 kb was sequenced, including 3 kb of upstream sequence. The predicted amino acid sequence showed closest similarity to plaice CYP1A1 (98%). Gene structure conformed to the seven exons and six introns common to previous CYP1A sequences, but intron lengths were not conserved. Concensus sequences corresponding to xenobiotic and other response elements as well as TATA, CAAT and GC boxes were identified. Upstream sequence (3.5 kb) including the first exon and intron up to the putative start codon were amplified via PCR and inserted upstream of the luciferase gene in a pGL3 reporter gene construct. The HepG2 mammalian hepatoma cell line was transiently co-transfected with the flounder CYP1A reporter gene construct and the pRL-CMV internal control construct. The maximal induction upon exposure to 100 nM 3-MC was 4.4-fold in comparison with carrier-treated cells. Use of deletion constructs resulted in loss of inducibility.     

Effects of the inhibitor ellipticine on cytochrome P450-reductase and cytochrome P450 (1A) function in hepatic microsomes of flounder (Platichthys flesus)

The effects of the mammalian inhibitor ellipticine (5,11-dimethyl-[6H]-pyrido[4,3b] carbazole) were examined in a mechanistic study of the cytochrome P450 monooxygenase system of control and β-naphthoflavone (βNF)-induced hepatic microsomes of Platichthys flesus. Ellipticine was indicated to bind to the haem moiety of cytochrome P450s (gave type II binding spectra) and to inhibit the transfer of electrons from both the hydrophobic binding site of cytochrome P450 reductase (P450R) to P450 (inhibited P450R reductase activity) and the hydrophilic binding site of P450R to soluble electron acceptors (inhibited NAD(P)H-cytochrome c reductase activity). No effect was seen on cytochrome b₅ reductase activity. Ellipticine inhibition indicated the involvement of (i) P450R (possibly also P450s) in NADPH- but not NADH- dependent hydroxyl radical production, and (ii) electron transfer and P450/P450R interaction in NADPH-dependent cytochrome P450 1A-catalysed monooxygenation (7-ethoxyresorufin O-deethylase activity and benzo(a)pyrene (BaP) metabolism). Differential effects of ellipticine on cumene hydroperoxide (CHP)-dependent BaP metabolism (P450 peroxidase activity) with CHP concentration indicated the existence of at least two forms of P450 with different substrate affinities for CHP, and different mechanisms of formation for protein adducts and free metabolites. Overall, the studies indicate the primary site of action of ellipticine in P. flesus is binding between Fe³⁺-P450 and P450R.     

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.     

Multi-technique approach to characterise the effects of cryopreservation on larval development of the Pacific oyster (Crassostrea gigas)

Cryopreservation experiments were conducted on D-stage larvae of the Pacific oyster (Crassostrea gigas) to investigate the effects of two cryoprotectant solutions and three cooling rates on larval development from 1 to 22 days post-fertilisation. Cryoprotectant solutions were made up to final concentrations (after 1:1 dilution with larvae) of 10% ethylene glycol, 1% polyvinylpyrrolidone and either 0.2 or 0.4 M trehalose. Three cooling rates (0.5, 1 and 2 °C min^?1 between ?10 and ?35 °C post-holding) were tested in an orthogonal design with the two cryoprotectants. Results indicate that control larvae out-performed all cryopreservation treatments for survival, feeding consumption and shell length parameters. However, larvae exposed to 0.4 M trehalose did considerably better than those exposed to 0.2 M trehalose, regardless of cooling rate conditions. Scanning electron and light microscopy observations were used to assess larval morphology and organogenesis, indicating that treatments with surviving larvae were morphologically and developmentally similar to control larvae.     

Seasonal variation in the reproductive activity and biochemical composition of the Pacific oyster (Crassostrea gigas) from the Marlborough Sounds,New Zealand

Reproductive cycles of the Pacific oyster Crassostrea gigas (Thunberg) from the Marlborough Sounds, New Zealand, were followed between June 1998 and January 2000. Histological examination of the gonad confirms an annual cycle with a winter inactive period followed by rapid gonad development and a single short spawning period. The population gonad index correlated with seawater temperature and changes in tissue dry weight, condition index, and biochemical components. In winter, few individuals with early gametogenic stages were present and rapid development of primary oocytes (diam. 11 μm) occurred during spring (September‐November). The developmental rate and the diameter of mature oocytes (37 μm) was similar for the 1998 and 1999 seasons. For a standard 110‐mm‐length oyster, maximal tissue body weight and condition index were recorded in December. Rapid weight loss in January was length dependent and was attributed to spawning. Temperature was the environmental variable which best correlated with the timing of gametogenesis. Food availability (phytoplankton biomass) may have been responsible for inter‐annual variations. The biochemical composition (% glycogen, lipid, protein) of separated gonad and somatic tissues were variable seasonally and annually. Gametogenesis (oocyte diameter) was associated with increased gonad protein and glycogen and a decrease in lipid concentrations. These changes are similar to those in Pacific oyster populations from other parts of the world.     

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

Feasibility of co-culture of the Australasian sea cucumber (Australostichopus mollis) with the Pacific oyster (Crassostrea gigas) in northern New Zealand

The Australasian sea cucumber (Australostichopus mollis) has attracted commercial attention for aquaculture development, partly due to its potential for co-culture with shellfish and finfish species. However, minimal attention has been given to the possibility of co-culturing this species with oysters. In this study we evaluated the growth of juvenile sea cucumbers (36.7 ± 0.9 g, wet weight) caged underneath Pacific oyster farms in northern New Zealand. Co-culture started at the end of the summer, and after 304 days the juveniles had doubled in size (79.8 ± 3.3 g, wet weight), but their subsequent growth appeared to be constrained by overstocking of the cages and summer water temperatures, reaching a carrying capacity of 720 g m^?2. Overall, the results of this study indicate that the co-culture of juvenile sea cucumbers with Pacific oysters is feasible, if sea cucumber losses are reduced (between 33% and 52% lost in this study) and careful attention is given to stocking rates and the water temperature regimes of oyster farms in order to maintain adequate growth rates.     

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.     

Alkoxyquinoline O-dealkylation: A new fluorimetric assay for hydrocarbon-induced cytochrome P450 in fish

The O-dealkylation of a new series of substrates, the alkoxyquinolines, provides a new easy fluorimetric assay for measuring the induction of cytochrome P450 by polycyclic aromatic hydrocarbons and drilling mud oil in fish. Expression of the results as a ratio between the inducible butyloxyquinoline O-dealkylation (BuQOD) activity and the non-inducible benzyloxyquinoline O-dealkylation (BeQOD) activity (i.e. the ‘BuQOD:-BeQOD quotient’) maximises the degree of induction measured and minimises the degree of inter-animal variation.     

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.