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.     

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].     

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.     

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。     

Evaluation of ecotoxicological effects of endocrine disrupters during a four-year survey of the Mediterranean population of swordfish (Xiphias gladius)

In this project we investigated the ecotoxicological effects of endocrine disrupters in a four-year survey of the Mediterranean population of swordfish (Xiphias gladius). In the Mediterranean environment, top predators, such as swordfish, accumulate high concentrations of polyhalogenated aromatic hydrocarbons (PHAHs) and toxic metals, potentially incurring high toxicological risk. The effects of organochlorines and trace elements (Hg, Cd and Pb) in 192 swordfish specimens, caught in the Strait of Messina, Sicily, Italy, were investigated using vitellogenin (Vtg), zona radiata proteins (Zrp) and CYP1A (BPMO, EROD) activities. Vtg and Zrp were found to be dramatically induced in some adult male specimens, suggesting that this species is highly exposed to estrogens in the Mediterranean Sea. A role of organochlorines in this induction phenomenon is suggested by the statistically significant correlations between Zrp in plasma and PCB concentrations in muscle (p<0.032) and Vtg in plasma and PCB concentrations in liver (p<0.034) of male specimens. Levels of trace elements in liver were in the following ranges: Hg 1–22, Cd 1–28 and Pb 0–1.6 ppm d.w. These data indicate potential reproductive alterations in large pelagic fish and suggest the need for continuous monitoring to avoid reductions in the population of this fish species of high commercial and ecological interest.     

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.     

In vitro mechanistic differences in benzo[a]pyrene–DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by ³²P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or β-naphthoflavone (ß-NF) or digestive glands from mussels. The β-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10⁸ nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the ³²P-postlabelled BaP 7,8-diol, 9,10-epoxide-N²-guanine adduct. Fewer adducts (P <0.05) were generated by BaP-induced microsomes (9.4–30.6 adducts per 10⁸ nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10⁸ nucleotides). Co-incubation with 500 μM α-naphthoflavone (α-NF) resulted in 97–99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.     

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%).     

The expression of CYP1A, vitellogenin and zona radiata proteins in Atlantic salmon (Salmo salar) after oral dosing with two commercial PBDE flame retardant mixtures: absence of short-term responses

The short-term effects of the commercial PBDE flame retardant mixtures Penta-BDE and Octa-BDE on the expression of cytochrome P450 1A (CYP1A), vitellogenin (Vtg) and zona radiata proteins (Zrp) were investigated in juvenile salmon (Salmo salar). For this purpose, groups of fish were dosed twice (oral intake at days 1 and 4) with 10 and 50 mg/kg body weight of both commercial mixtures. The fishes were sacrificed at day 7 (n=5 for each group) and 14 (n=6 for each group), and blood, liver, fillet, and brain were collected. Blanks and positive controls were also part of the experiment. The expressions of Vtg, Zrp, and CYP1A were measured with several techniques (EROD, ELISA, Western, Northern and Slot Blot). The values in the groups of fish treated with Penta-BDE or Octa-BDE did not significantly differ from the reference group for any of the parameters tested. In contrast, the positive control groups treated with estradiol-17β for Vtg and Zrp expression, and β-naphthoflavone for CYP1A expression did show a significant response, indicating the potential sensitivity of the fishes for the parameters measured. Since the results of the chemical analyses showed concentrations of a number of PBDE congeners in liver, fillet, and brain that were about three orders of magnitude above those of fish from the North Sea, it is concluded that the short-term toxicity of both commercial PBDE mixtures for these endpoints was low.     

Effects of β-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to β-naphthoflavone (β-NF, 66 mg/kg, i.p.) elicited a 7–9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to β-NF, and generally tracked the minimal changes observed in GST–CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by β-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to β-NF.     

Quantification of cytochrome P4501A1 and catalytic activities in liver microsomes of isosafrol- and β-Naphthoflavone-treated rainbow trout (Oncorhynchus mykiss)

The effects of isosafrol (ISF) or β-naphthoflavone (βNF) treatments on cytochrome P450 (P450) levels in rainbow trout liver were investigated using immunochemical and catalytic techniques. The discrepancies in catalytic activities and ELISA quantification of rainbow trout P4501A1 protein levels between ISF- and βNF-treated fish indicate that important differences exist between the responses induced by βNF and ISF treatments in the rainbow trout liver.     

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.     

The DNA de-methylating agent 5-azacytidine does not restore CYP1A induction in PCB resistant Newark Bay killifish (Fundulus heteroclitus)

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 μ(micro)M) with or without 0.2 μ(micro)g/l of the CYP1A inducer 3,3^′,4,4^′,5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.     

Development of two novel CYP-antibodies and their use in a PCB exposure experiment with Mytilus edulis

In an attempt to learn more about the cytochrome P450 (CYP) system of mussels, we used protein databases and alignment software to extract highly conserved CYP sequences. From these alignments synthetic peptides were produced and used for rabbit immunisation, which yielded polyclonal antibodies against the CYP families 2 and 4. The antibodies were evaluated with Western Blot and ELISA assays, using digestive gland microsomal samples from the mussel Mytilus edulis. Western Blots revealed immunoreactions for both antibodies. The anti-CYP2 sequence rendered one major immunopositive protein of ≈49 kDa size, and weak signals for proteins of ≈41 and 56 kDa size. The anti-CYP4 sequence rendered two major bands of ≈56 and 59 kDa size, and also a weak immunoreaction with a protein of ≈43 kDa size. ELISA rendered only weak signals even with a 1:50 dilution of IgG-purified serum. A 10-day exposure to Aroclor 1254 did not appear to affect any of the immunopositive proteins, while total PCBs in soft bodies increased from 14–40 ng/g DW in controls to 373–638 ng/g DW in exposed mussels.