Comparative endocrine disruptive effects of contaminants in ringed seals (Phoca hispida) from Svalbard and the Baltic Sea

We investigated variables related to thyroid, vitamin A and calcitriol homeostasis, immune function and tumour development in ringed seals (Phoca hispida) from the polluted Baltic Sea and a less polluted reference location at Svalbard, Norway. We also examined the relationships between the biological variables and the concentrations of persistent organic pollutants (POPs) and their hydroxylated (OH) metabolites. Our data show higher plasma concentrations of free triiodothyronine (T3), and ratios of free and total T3 in Baltic seals as compared to Svalbard seals. Baltic seals had also higher hepatic mRNA expressions of deiodinase-I, thyroid hormone receptor β, retinoic acid receptor α, growth hormone receptor and interleukin-1β compared to Svalbard seals. Levels of plasma retinol were lower in the Baltic seals as compared to Svalbard seals. No geographical difference was observed for other thyroid hormone levels and hepatic retinoid levels. Ratios of free and total T3 were positively correlated to OH-POPs in plasma. The results of the present study suggest that endocrine homeostasis may be affected by contaminant and metabolite exposure in the Baltic ringed seals with respect to circulating hormones and retinol and hepatic mRNA expressions. In addition, OH-POPs may putatively produce the disruption of thyroid hormone transport in plasma.     

Microsatellite and mtDNA analysis of the population structure of grey seals (Halichoerus grypus) from three breeding areas in the Baltic Sea

The growing number of grey seals in the Baltic Sea has led to a dramatic increase in interactions between seals and fisheries. The conflict has become such a problem that hunting was introduced in Finland in 1998 and the Swedish Environment Protection Agency recommended a cull of grey seals starting in 2001. Culling has been implemented despite the lack of data on population structure. Low levels of migration between regions would mean that intensive culling in specific geographic areas would have disproportionate effects on local population structure and genetic diversity. We used eight microsatellite loci and a 489 bp section of the mtDNA control region to examine the genetic variability and differentiation between three breeding sites in the Baltic Sea and two in the UK. We found high levels of genetic variability in all sampled Baltic groups for both the microsatellites and the control region. There were highly significant differences in microsatellite allele frequencies between all three Baltic breeding sites and between the Baltic sites and the UK sites. However, there were no significant differences in mtDNA control region haplotypes between the Baltic sites. This genetic substructure of the Baltic grey seal populations should be taken into consideration when managing the seal population to prevent the hunting regime from having an adverse effect on genetic diversity by setting hunting quotas separately for the different subpopulations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phylogenetic Analysis of the Cytochrome P450 3 (CYP3) Gene Family

Cytochrome P450 genes (CYP) constitute a superfamily with members known from the Bacteria, Archaea, and Eukarya. The CYP3 gene family includes the CYP3A and CYP3B subfamilies. Members of the CYP3A subfamily represent the dominant CYP forms expressed in the digestive and respiratory tracts of vertebrates. The CYP3A enzymes metabolize a wide variety of chemically diverse lipophilic organic compounds. To understand vertebrate CYP3 diversity better, we determined the killifish (Fundulus heteroclitus) CYP3A30 and CYP3A56 and the ball python (Python regius) CYP3A42 sequences. We performed phylogenetic analyses of 45 vertebrate CYP3 amino acid sequences using a Bayesian approach. Our analyses indicate that teleost, diapsid, and mammalian CYP3A genes have undergone independent diversification and that the ancestral vertebrate genome contained a single CYP3A gene. Most CYP3A diversity is the product of recent gene duplication events. There is strong support for placement of the guinea pig CYP3A genes within the rodent CYP3A diversification. The rat, mouse, and hamster CYP3A genes are mixed among several rodent CYP3A subclades, indicative of a complex history involving speciation and gene duplication. Phylogenetic analyses suggest two CYP3A gene duplication events early in rodent history, with the rat CYP3A9 and mouse Cyp3a13 clade having a sister relationship to all other rodent CYP3A genes. In primate history, the human CYP3A43 gene appears to have a sister relationship to all other known primate CYP3A genes. Other, more recent gene duplications are hypothesized to have occurred independently within the human, pig, rat, mouse, guinea pig, and fish genomes. Functional analyses suggest that gene duplication is strongly tied to acquisition of new function and that convergent evolution of CYP3A function may be frequent among independent gene copies. Current address (Rachel L. Cox): Laboratory of Aquatic Biomedicine, Marine Biology Laboratory, Woods Hole, MA 02543, USA     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Active-site residues move independently from the rest of the protein in a 200 ns molecular dynamics simulation of cytochrome P450 CYP119

The conformational dynamics of cytochrome P450 enzymes are critical to their catalytic activity. In this study, the correlated motion between residues in a 200 ns molecular dynamics trajectory of the thermophilic CYP119 was analyzed to parse out conformational relationships. Residues that are structurally related, for example residues within a helix, generally have highly correlated motion. In addition, clusters of non-adjacent residues that show correlated motion (“hot spots”) are seen in various regions, including at the base of the F and G helices that make up the most dynamic region of the enzyme. A modified k-means algorithm that clusters residues based on their correlated motion indicates that functionally related residues are in the same cluster (e.g., the catalytic threonines and the heme). Tightly coupled clusters form a solvent-exposed “shell” around the enzyme, whereas less coupling between clusters is seen in regions that are critical to ligand interactions, redox partner interactions, and catalysis. Most notably, we find that residues in the active site move independently from the rest of the enzyme, effectively insulating the catalytic machinery from other regions of the protein.     

Functional interpretation and structural insights of Arabidopsis lyrata cytochrome P450 CYP71A13 involved in auxin synthesis

Cytochrome P450 CYP71A13 of Arabidopsis lyrata is a heme protein involved in biosynthesis of indole-3-acetonitrile which leads to the formation of indolyl-3-acetic acid. It catalyzes a unique reaction: formation of a carbon-nitrogen triple bond and dehydration of indolyl-3-acetaldoxime. Homology model of this 57 kDa polypeptide revealed that the heme existed between H-helix and J- helix in the hydrophobic pocket, although both helixes are involved in catalytic activity, where Gly305 and Thr308, 311 of H- helix were involved in its stabilization. The substrate indole-3-acetaldoxime was tightly fitted into the substrate pocket with the aromatic ring being surrounded by amino acid residues creating a hydrophobic environment. The smaller size of the substrate binding pocket in cytochrome P450 CYP71A13 was due to the bulkiness of the two amino acid residues Phe182 and Trp315 pointing into the substrate binding cavity. The apparent role of the heme in cytochrome P450 CYP71A13 was to tether the substrate in the catalysis by indole-3-acetaldoxime dehydratase. Since the crystal structure of cytochrome P450 CYP71A13 has not yet been solved, the modeled structure revealed mechanism of substrate recognition and catalysis.     

Characterization of cytochrome P450 monooxygenases isolated from trichome enriched fraction of Artemisia annua L. leaf

CYPs have major role in the biosynthesis and modification of secondary metabolites. Predicting the possible involvement of CYPs in secondary metabolism, 20 partial sequences were amplified from the cDNA of trichome enriched tissue of Artemisia annua. Seven CYPs were converted to full length and assigned to different families based on sequence homology. These were co-expressed with CPR in Saccharomyces cerevisiae and microsome fractions were assayed for conversion of sesquiterpenes, phenols and fatty acid substrates. CIM_CYP02(c73) and CIM_CYP05(c81) converted trans-cinnamic acid to p-coumaric acid; and capric acid, lauric acid to their hydroxylated products, respectively. Higher expression of CIM_CYP71AV1, CIM_CYP03(c72a), CIM_CYP06(c72b), CIM_CYP02(c73) and CIM_CYP04(c83) was observed in the mature leaf, whereas expression of CIM_CYP05(c81) was more in the seedling. CIM_CYP71AV1, CIM_CYP02(c73) and CIM_CYP04(c83) expressed more in the flower bud compared to the leaf, with minor expression in stem. All CYPs' expression increased progressively with time after wounding except for CIM_CYP07(c92). These results relate involvement of CIM_CYP02(c73) to phenyl-propanoid metabolism in the leaf and CIM_CYP05(c81) to fatty acid metabolism in the seedling. Expression of CIM_CYP71AV1 and CIM_CYP02(c73) significantly increased when sprayed with trans-cinnamic acid indicating a relationship between phenylpropanoid and artemisinic acid pathways.     

Synthesis and evaluation of inhibitors of cytochrome P450 3A (CYP3A) for pharmacokinetic enhancement of drugs

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2–P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.     

Functional differences in the cytochrome P450 1 family enzymes from Zebrafish (Danio rerio) using heterologously expressed proteins

Mammalian cytochrome P450 1 (CYP1) genes are well characterized, but in other vertebrates only the functions of CYP1A genes have been well studied. We determined the catalytic activity of zebrafish CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1 proteins using 11 fluorometric substrates and benzo[a]pyrene (BaP). The resorufin-based substrates, 7-ethoxyresorufin, 7-methoxyresorufin, and 7-benzyloxyresorufin, were well metabolized by all CYP1s except CYP1D1. CYP1A metabolized nearly all substrates tested, although rates for non-resorufin substrates were typically lower than resorufin-based substrates. Zebrafish CYP1s did not metabolize 7-benzyloxyquinoline, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin or 7-methoxy-4-(aminomethyl)-coumarin. CYP1B1 and CYP1C2 had the highest rates of BaP metabolism. 3-Hydroxy-BaP was a prominent metabolite for all but CYP1D1. CYP1A showed broad specificity and had the highest metabolic rates for nearly all substrates. CYP1C1 and CYP1C2 had similar substrate specificity. CYP1D1 had very low activities for all substrates except BaP, and a different regioselectivity for BaP, suggesting that CYP1D1 function may be different from other CYP1s.     

High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy: II. Validation of a direct injection/on-line guard cartridge extraction–tandem mass spectrometry method for CYP2D6 inhibition assessment

A highly efficient direct injection/on-line guard cartridge extraction–tandem mass spectrometry (DI/GCE–MS–MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 2D6 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE–MS–MS analysis. Due to the novel use of an extremely short C₁₈ guard cartridge, this method exhibits several advantages, such as no sample preparation, excellent on-line extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI/GCE–MS–MS method demonstrates acceptable accuracy and precision for the quantification of dextrorphan, a marker metabolite of dextromethorphan mediated by CYP2D6, in microsomal incubations. The CYP2D6 inhibition assay has been validated using quinidine as a known selective inhibitor of the isoform. The IC₅₀ value (0.20 μM) measured by the new method is in good agreement with the literature value (0.22 μM).     

The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution.     

Eicosapentaenoic acid metabolism by cytochrome P450 enzymes of the CYP2C subfamily

CYP2C enzymes epoxidize arachidonic acid (AA) to metabolites involved in the regulation of vascular and renal function. We tested the hypothesis that eicosapentaenoic acid (EPA), a n-3 polyunsaturated fatty acid, may serve as an alternative substrate. Human CYP2C8 and CYP2C9, as well as rat CYP2C11 and CYP2C23, were co-expressed with NADPH-CYP reductase in a baculovirus/insect cell system. The recombinant enzymes showed high EPA and AA epoxygenase activities and the catalytic efficiencies were almost equal comparing the two substrates. The 17,18-double bond was the preferred site of EPA epoxidation by CYPs 2C8, 2C11, and 2C23. 17(R),18(S)-Epoxyeicosatetraenoic acid was produced with an optical purity of about 70% by CYPs 2C9, 2C11, and 2C23 whereas CYP2C8 showed the opposite enantioselectivity. These results demonstrate that EPA is an efficient substrate of CYP2C enzymes and suggest that n-3 PUFA-rich diets may shift the CYP2C-dependent generation of physiologically active eicosanoids from AA- to EPA-derived metabolites.     

细胞色素P450还原酶与CYP17的共表达及其功能分析*

17α-羟基黄体酮(17α-OH-PROG)是甾体激素类药物的关键中间体,其生物合成主要由细胞色素单加氧酶(CYP17)催化生成。在此过程中,细胞色素 P450还原酶(cytochrome P450 reductase,CPR)作为细胞色素P450 酶电子传递链的重要组成部分,直接影响CYP17的催化效率。为研究不同来源CPR与17α-羟化酶的适配性,首先以人源17α-羟化酶作为研究对象,构建了表达质粒pPIC3.5k-hCYP17,获得了重组毕赤酵母菌株。其次筛选获得3种不同来源CPR,构建了表达质粒 pPICZX-CPR,获得17α-羟化酶与CPR共表达菌株,并在毕赤酵母中进行转化实验,对转化产物进行薄层色谱(TLC)和高效液相色谱(HPLC)分析。结果显示,重组菌株具有17α-羟化酶活性,能够催化黄体酮生成目标产物17α-OH-PROG 以及副产物16α-羟基黄体酮(16α-OH-PROG)。不同来源的CPR与17α-羟化酶共表达与仅表达17α-羟化酶的产率相比均有所提高,其中hCPR-CYP17共表达菌株表现出最高的转化水平,17α-OH-PROG产率提高42%。上述结果表明:17α-羟化酶基因与CPR共表达能够提高其黄体酮17α-羟基化水平。为甾体黄体酮17α-羟基化的生物催化研究提供思路,对甾体药物的工业生产具有重要意义。     

南沙链霉菌来源细胞色素P450酶CYP154C34的表征及生物转化

【目的】P450酶作为一种多功能生物催化剂,可在温和条件下高区域和立体选择性地催化复杂化合物中未活化的C-H键,因此P450酶在化工原料合成、环境污染物降解及药物合成等领域都具有重要作用。本文对南沙链霉菌基因组中的一个新颖的P450酶CYP154C34进行研究,通过构建异源表达和全细胞生物转化重组菌探究其功能。【方法】构建2种全细胞生物转化BL21(DE3)重组菌(含p ET28a-CYP154C34-RhFRED和pET28a-CYP154C34+pACYCDuet-Pdx/PdR)和1种异源表达BL21(DE3)重组菌(含pET28a-CYP154C34)。通过全细胞生物转化的方式筛选底物,分析催化功能及产物结构。比较2种全细胞生物转化重组菌和体外酶反应对底物的转化率。分析CYP154C34和不同底物及底物类似物的亲和力。【结果】通过底物筛选和产物鉴定发现CYP154C34可催化包括孕酮、睾酮、雄烯二酮在内的9种甾体化合物16α位羟基化。通过2种不同还原伴侣的全细胞体系及体外酶反应对底物转化率的比较,发现含有pET28a-CYP154C34-RhFRED的BL21(DE3)重组菌的...     

Chlorzoxazone hydroxylation in microsomes and hepatocytes from cytochrome P450 oxidoreductase‐null mice

Previous studies have demonstrated that the NADH‐dependent cytochrome b₅ electron transfer pathway can support some cytochrome P450 monooxygenases in vitro in the absence of their normal redox partner, NADPH‐cytochrome P450 oxidoreductase. However, the ability of this pathway to support P450 activity in whole cells and in vivo remains unresolved. To address this question, liver microsomes and hepatocytes were prepared from hepatic cytochrome P450 oxidoreductase‐null mice and chlorzoxazone hydroxylation, a reaction catalyzed primarily by cytochrome P450 2E1, was evaluated. As expected, NADPH‐supported chlorzoxazone hydroxylation was absent in liver microsomes from oxidoreductase‐null mice, whereas NADH‐supported activity was about twofold higher than that found in normal (wild‐type) liver microsomes. This greater activity in oxidoreductase‐null microsomes could be attributed to the fourfold higher level of CYP2E1 and 1.4‐fold higher level of cytochrome b₅. Chlorzoxazone hydroxylation in hepatocytes from oxidoreductase‐null mice was about 5% of that in hepatocytes from wild‐type mice and matched the results obtained with wild‐type microsomes, where activity obtained with NADH was about 5% of that obtained when both NADH and NADPH were included in the reaction mixture. These results argue that the cytochrome b₅ electron transfer pathway can support a low but measurable level of CYP2E1 activity under physiological conditions. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:357–363, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20299     

Structural characterization of CYP165D3, a cytochrome P450 involved in phenolic coupling in teicoplanin biosynthesis

Teicoplanin is a glycopeptide antibiotic with activity against Gram-positive bacteria and remains one of the last lines of clinical defense against certain bacterial infections. We have cloned, expressed, and purified the cytochrome P450 OxyE (CYP165D3) from the teicoplanin biosynthetic gene cluster of Actinoplanes teichomyceticus, which is responsible for the phenolic coupling of the aromatic side chains of the first and third peptide residues in the teicoplanin peptide. The crystal structure of OxyE has been determined to 2.5 Å resolution, revealing the probable binding surface for the carrier protein substrate and an extension of the active site into a pocket located above the β-1 sheet. The binding of potential substrates to OxyE shows that peptidyl carrier protein-bound linear peptides bind to OxyE, albeit with low affinity in the absence of a phenolic cross-link that should normally be installed by another Oxy protein in the teicoplanin biosynthetic pathway. This result indicates that the carrier protein alone is not sufficient for tight substrate binding to OxyE and that the Oxy proteins sense the structure of the bound peptide in addition to the presence of the carrier protein, a feature distinct from other carrier protein/P450 systems.