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.     

Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos

We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus lambdaGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced approximately 7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFkappaB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 3'-UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms' c-fos genes. We propose its potential use in ecotoxicology.     

Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos

We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus λGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced ≈7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFκB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 3^′-UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms' c-fos genes. We propose its potential use in ecotoxicology.     

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.     

Genomic cloning and expression of vitellogenin gene from the self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae)

We cloned the vitellogenin gene from the self-fertilizing fish Rivulus marmoratus, and sequenced 12,326 bp. The number of exons of R. marmoratus and rainbow trout vitellogenin genes were different, and also the splicing junctions are different throughout most of the exons and introns but the amino acid similarity of R. marmoratus vitellogenin gene to other species was rather high. In promoter region of R. marmoratus vitellogenin gene, there were several E2 binding sites and the estrogen response element (ERE). We discuss here the gene structure and expression of R. marmoratus vitellogenin gene.     

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.     

Genomic cloning and expression of vitellogenin gene from the self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae)

We cloned the vitellogenin gene from the self-fertilizing fish Rivulus marmoratus, and sequenced 12,326 bp. The number of exons of R. marmoratus and rainbow trout vitellogenin genes were different, and also the splicing junctions are different throughout most of the exons and introns but the amino acid similarity of R. marmoratus vitellogenin gene to other species was rather high. In promoter region of R. marmoratus vitellogenin gene, there were several E2 binding sites and the estrogen response element (ERE). We discuss here the gene structure and expression of R. marmoratus vitellogenin gene.     

雌牙鲆CYP19A基因启动子“CpG”岛区的单核苷酸多态性及与繁殖性能的关联分析

芳香化酶基因(CYP19)编码的芳香化酶是雄激素转化为雌激素的关键酶,在鱼类性腺发育及繁殖内分泌过程中起着重要的作用。本研究采用聚合酶链式反应-单链构象多态(PCR-SSCP)和放射性免疫方法(RAI)对61尾雌牙鲆进行了CYP19A基因启动子CpG岛区多态性检测,并将其与繁殖性能进行了关联分析。CpG岛区存在3种类型的基因型(pattern1、pattern2、pattern3),经测序发现CpG岛区有以下突变:T-1927插入或缺失、C-1899插入或缺失、T-1772C、C-1766T、TGTCAAC-1752~-1744插入或缺失(11个SNPs连锁突变)。方差分析结果表明:此位点对雌二醇(E2)水平的效应达到显著(P<0.05)水平,经多重比较分析,具有pattern1基因型的个体的雌激素(E2)水平显著低于pattern2和pattern3基因型个体的雌激素水平(P<0.05),同时pattern1基因型的个体的mRNA表达量显著低于pattern2和pattern3基因型个体的mRNA表达量(P<0.05)。以上结果表明,CYP19A基因调控区甲基化位点突变可能导致芳香化酶的表达量,进而影响牙鲆繁殖内分泌过程;同时也阐明了这些与牙鲆繁殖性状相关的SNPs可以用于生产实践中进行标记辅助选择具有优良繁殖性能的牙鲆。     

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.     

Induction of Cyp1A in Japanese medaka exposed to carbonaceous pitches via water

The objective of these studies was to characterize hepatic Cyp1A induction by complex carbonaceous pitches both in Japanese medaka exposed via aquarium water and in cultured fish liver cells. Carbonaceous pitches were extracted with DMSO and added to aquaria water of medaka or to cultured medaka or trout liver cells, and CyplA induction was assessed by EROD assay. When medaka were exposed to different carbonaceous pitches, EROD activity was induced to different extents, and increased EROD induction was associated with increasing temperature of hydrogenation. EROD induction in cultured fish liver cells was somewhat less sensitive than that observed in vivo. These studies indicate CyplA induction in medaka can be used to detect compounds, such as polyaromatic hydrocarbons, in complex samples and could be used as a biomarker for the presence of these compounds in the environment.     

Analysis of medaka cytochrome P450 3A homotropic and heterotropic cooperativity

We have previously demonstrated that medaka CYP3A is associated with metabolism of several endobiotics including steroids and bile acids. In this study, we demonstrate that medaka CYP3A catalysis exhibits unusual kinetic behaviors consistent with allosteric interaction which cannot be described by hyperbolic kinetic models. Using 7-benzyloxy-4-(trifluoromethyl)-coumarin (BFC) and nonylphenol as CYP3A substrates, we describe both homotropic and heterotropic cooperative interactions. Given the role of CYP3A in maintaining the homeostatic balance for numerous endobiotics, enzymatic activation/inhibition by endocrine disruptors (EDCs) represents a putative (non-genomic) mechanism for endocrine disruption.     

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.     

菲、芘、苯并(a)芘单一暴露及分别与α-萘黄酮(ANF)联合暴露对海水青鳉(Oryzias melastigma)胚胎发育毒性效应的比较研究

为了解和探讨3~5环PAHs对海水鱼类胚胎发育的毒性及作用方式,比较研究了菲(phenanthrene,Phe)、芘(pyrene,Py)、苯并(a)芘(benzo(a)pyrene,BaP)单一暴露和三者各自与α-萘黄酮(α-naphthoflavone,ANF)联合暴露对海水青鳉(marine medaka, Oryzias melastigma)胚胎发育的毒性效应。胚胎体内EROD活性、发育畸形、孵化率和心律等毒性指标被测定,结果显示:Phe,Py和BaP对海水青鳉胚胎体内EROD活性的诱导能力大小为BaP>Py>Phe,各化合物对EROD诱导与发育畸形之间的关系较为复杂,除Phe所引起的EROD诱导与畸形指数之间呈显著相关(r=0.95,p=0.015)外,Py和BaP均无相关性;在100 μg/dm3 ANF影响下,CYP1A活性诱导被抑制,但胚胎发育的畸形指数被显著提高,ANF分别与Phe,Py和BaP的联合暴露对胚胎发育呈潜在的协同作用。本文研究初步表明,3~5环PAHs化合物对海水青鳉胚胎发育的毒性作用方式可能不同;CYP1A活性抑制在PAHs混合物对海水青鳉胚胎发育的毒性作用过程中未起到缓解毒性的作用,CYP1A抑制剂与PAH型CYP1A诱导剂的混合物对鱼类胚胎发育具有潜在的协同毒性作用,现有的PAHs混合物毒性风险评价方法可能低估了实际环境中PAHs的风险;海水青鳉早期生活阶段的心脏发育对PAHs混合物暴露较为敏感,可推荐其作为生物标志物指示PAHs或溢油污染。     

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.