Deregulation of the oxidative cascade of poly-unsaturated fatty acids (PUFAs) has been associated with several cancers, including chronic lymphocytic leukemia (B-CLL). Leukotriene B4 (LTB4), a metabolite of arachidonic acid (AA), is produced by B-CLL and contributes to their survival. The aim of the present study was to analyze the activity of the oxidative cascade of PUFAs in B-CLL. Purified B cells from patients and normal B CD5 positive cells were subjected to flow cytometry, Western-blot and RT-qPCR analyses. LTB4 plasma and intracellular concentrations were determined by ELISA. Our results showed that aggressive B-CLL tumor cells, i.e. cells with an annual proliferation index above 2, over-expressed calcium-dependent and calcium-independent phospholipases A2 (cPLA2-alpha and iPLA2-beta, respectively), 5-lipoxygenase (5LOX) and leukotriene A4 hydroxylase (LTA4H). Intracellular LTB4 levels were lower in the most aggressive cells than in cells with a smaller proliferation index, despite equivalent plasma levels, and lower expression of cytochrome P450 4F3A (CYP4F3A), one major enzyme involved in LTB4 inactivation. Since BLT2, a LTB4 membrane receptor was also more often expressed on aggressive tumor cells, and since a BLT2 inhibitor significantly impaired B-CLL viability in vitro, we propose that LTB4 was efficiently trapped onto BLT2 present on aggressive tumors, thereby eliciting an autocrine response. Taken together our results demonstrate a major deregulation of the pathway leading to LTB4 synthesis and degradation in B-CLL cells, and provide a framework for understanding how these modifications promote cell survival and proliferation, especially in the most aggressive BCLL.

<正>研究人员试图运用功能和生物学方法确定环氧花生四烯酸、一氧化氮/活性氧平衡及内皮素1在原发性高血压患者传导性动脉内皮功能紊乱中所起的作用。方法与结果:研究对象为未经治疗的原发性高血压患者28例和正常血压者30名,测定其由于手部皮肤加

In young healthy subjects, epoxyeicosatrienoic acids synthesized by endothelial cytochrome P450 epoxygenases maintain basal conduit artery diameter during altered NO availability. Whether this compensatory mechanism is effective during essential hypertension is unknown. Radial artery diameter, blood flow, and mean wall shear stress were determined in 14 nontreated essential hypertensive patients and 14 normotensive control subjects during 8 minutes of brachial infusion for inhibitors of cytochrome P450 epoxygenases (fluconazole, 0.4 µmol/min) and NO synthase (N(G)-monomethyl-L-arginine, 8 µmol/min) alone and in combination. In controls, the radial artery diameter was reduced by fluconazole (-0.034 ± 0.012 mm) and N(G)-monomethyl-L-arginine (-0.037 ± 0.010 mm) and to a larger extent by their combination (-0.137 ± 0.011 mm), demonstrating a synergic effect. In contrast, the radial diameter in hypertensive patients was not affected by fluconazole (0.010 ± 0.014 mm) but was reduced by N(G)-monomethyl-L-arginine (-0.091 ± 0.008 mm) to a larger extent than in controls. In parallel, N(G)-monomethyl-L-arginine decreased local plasma nitrite to a lesser extent in hypertensive patients (-14 ± 5 nmol/L) than in controls (-50 ± 10 nmol/L). Moreover, the addition of fluconazole to N(G)-monomethyl-L-arginine did not further decrease radial diameter in patients (-0.086 ± 0.011 mm). Accordingly, fluconazole significantly decreased local epoxyeicosatrienoic acid plasma level in controls (-2.0 ± 0.6 ng/mL) but not in patients (-0.9 ± 0.4 ng/mL). Inhibitors effects on blood flow and endothelium-independent dilatation to sodium nitroprusside were similar between groups. These results show that, in contrast to normotensive subjects, epoxyeicosatrienoic acids did not contribute to the regulation of basal conduit artery diameter and did not compensate for altered NO availability to maintain this diameter in essential hypertensive patients.

Cytochrome P450 4F3 (CYP4F3), originally identified as one of the leukotriene B4 ω-hydroxylases, belongs to a CYP gene family that comprises several members, which participate in the metabolism of various endobiotics, as well as some xenobiotics. The CYP4F gene family is clustered in a 0.5-Mb stretch of genomic DNA on the p13 region of chromosome 19. Apart from the ω-hydroxylation of leukotriene B4 and prostaglandins, CYP4F3 is the main catalyst in the oxidation of fatty acid epoxides. CYP4F3 expression results from the synthesis of two distinct enzymes, CYP4F3A and CYP4F3B, which originate from the alternative splicing of a single pre-mRNA precursor molecule. Remarkably, the selection of either isoform is part of a tissue-specific control through which CYP3F3A is mostly expressed in leukocytes and CYP4F3B mostly in the liver. Recently, CYP4F3 single nucleotide polymorphisms have been incriminated in the onset of pathologies, including celiac or Crohn's diseases. Although much has been discovered in the regulation and function of CYP4F2, the closest CYP4F subfamily member, analyses of CYP4F3 enzymes lag somewhat behind in the field of our knowledge. In this short review, emphasis will be placed on the regulation and the functional roles of human CYP4F3.

Supplies of marine fish oils are limited, and continued growth in aquaculture production dictates that lipid substitutes in fish diets must be used without compromising fish health and product quality. In this study, the total substitution of a fish meal and fish oil by a blend of vegetable meals (corn, soybean, wheat and lupin) and linseed oil in the diet of European sea bass (Dicentrachus labrax) was investigated. Two groups of European sea bass were fed with fish diet (FD) or vegetable diet (VD) for 9months. VD, totally deprived of eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), revealed a nutritional deficiency and affected growth performance. Whilst VD induced a significant increase in fatty acid desaturase 2 (FADS2) and sterol binding regulatory element-binding protein 1 (SREBP-1) mRNA levels, the desaturation rate of [1-(14)C]18:3n-3 into [1-(14)C]18:4n-3, analysed in microsomal preparations using HPLC method, did not show an upregulation of FADS2 activities in liver and intestine of fish fed VD. Moreover Western-blot analysis did not revealed any significant difference of FADS2 protein amount between the two dietary groups. These data demonstrate that sea bass exhibits a desaturase (FADS2) activity whatever their diet, but a post-transcriptional regulation of fads2 RNA prevents an increase of enzyme in fish fed a HUFA-free diet. This led to a lower fish growth and poor muscle HUFA content.

Cytochromes P450 (CYPs) metabolize polyunsaturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., omega6 in AA or omega3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefin of AA (omega6), EPA (omega3), or DHA (omega3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs.

Human CYP450 omega-hydroxylases of the CYP4 family are known to convert arachidonic acid (AA) to its metabolite 20-hydroxyeicosatetraenoic acid (20-HETE). This study deals with hydroxylations of four PUFAs, eicosatrienoic acid (ETA), AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) by either human recombinant CYP4s enzymes or human liver microsomal preparations. CYP4F3A and CYP4F3B were the most efficient omega-hydroxylases of these PUFAs. Moreover, the differences in the number of unsaturations of ETA, AA, and EPA allowed us to demonstrate a rise in the metabolic rate of hydroxylation when the double bond in 14-15 or 17-18 was missing. With the CYP4F enzymes, the main pathway was always the omega-hydroxylation of PUFAs, whereas it was the (omega-1)-hydroxylation with CYP1A1, CYP2C19, and CYP2E1. Finally, we demonstrated that the omega9 and omega3 PUFAs (ETA, EPA, and DHA) could all be used as alternative substrates in AA metabolism by human CYP4F2 and -4F3B. Thus, they decreased the ability of these enzymes to convert AA to 20-HETE. However, although ETA was the most hydroxylated substrate, EPA and DHA were the most potent inhibitors of the conversion of AA to 20-HETE. These findings suggest that some physiological effects of omega3 FAs could partly result from a shift in the generation of active hydroxylated metabolites of AA through a CYP-mediated catalysis.

The regulation of the human liver-specific cytochrome P450 4F3B (CYP4F3B) isoform, a splice variant of the CYP4F3 gene with strong substrate specificity for long chain fatty acids, is yet an unsolved question. This report provides the first evidence that CYP4F3B is uniquely induced by prostaglandin A(1) (PGA(1)) in human hepatocyte-like HepaRG cells and leads to the synthesis of 20-hydroxy-eicosatetraenoic acids (HETEs). Real time PCR, immunoblot analysis with a specific antipeptide antibody, and determination of fatty acid omega-hydroxylase activity demonstrate that PGA(1) treatment strongly increases expression of CYP4F3B. This induction drives the production of 20-HETE (19-fold increase). SiRNA-mediated-silencing of CYP4F3 suppresses both 20-HETE synthesis and PGA(1) induced 20-HETE production. Taken together, these results provide evidence that CYP4F3B is the key enzyme to produce 20-HETE by omega-hydroxylation of arachidonic acid in liver cells. Since 20-HETE is a potent activator of PPAR alpha and an important inflammatory mediator, CYP4F3B may exert important functions in lipid homeostasis and in inflammatory diseases.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid involved in the regulation of vascular tone. Despite the importance of EETs in a variety of physiological effects, few methods have been developed to quantify them in human blood. This led us to develop a method by GC/MS with negative ion chemical ionization. As EETs are primarily located in phospholipids, red blood cells (RBCs) and plasma phospholipids were hydrolyzed with phospholipase A(2) after a solid phase extraction. Then, EETs were derivatized as pentafluorobenzyl esters, and [(2)H(8)]-arachidonic acid was used as internal standard for quantification. EETs were found to be at concentrations of 106+/-37ng mL(-1) in plasma and 33.4+/-8.5 ng/10(9) RBCs (mean+/-S.D.) in 10 healthy volunteers. Their amount in RBCs was 3-fold that in plasma; both parameters proved to be well correlated.

Epoxidation and hydroxylation of arachidonic acid (AA) are both catalyzed by cytochromes P450s (CYPs). The oxidized metabolites are known to be involved in the regulation of vascular tone and renal function. By using a panel of 15 human recombinant CYPs, this study demonstrates that other polyunsaturated long-chain fatty acids (PUFA-LC), especially the ω3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are also epoxidised. The regioselectivity of epoxidation of four PUFA-LC by CYPs was investigated. Among the several CYPs tested, CYP2C9/2C19 and 1A2 were the most efficient in EPA and DHA epoxidations. It ensued that 10μM of these two ω3 fatty acids decreased by more than 80% and 60%, respectively, the formation by CYP2C9 of AA-epoxidised derivatives. These findings suggest that some physiological effects of ω3 fatty acids may be due to a shift in the generation of active epoxidised metabolites of AA through CYP-mediated catalysis.

Despite the implication of polyunsaturated fatty acid monoepoxides in a large panel of biological effects, few methods allowing their separation in a single run are available. We describe here a simple method based on reversed-phase ion-pair high-performance liquid chromatography (RP-HPLC) and developed to successfully separate the various monoepoxides of eicosatrienoic, arachidonic, eicosapentaenoic and docosahexaenoic acids. These compounds were easily identified by liquid chromatography–mass spectrometry (LC–MS) with atmospheric pressure chemical ionisation owing to the volatility of counter-ion species. Compared to established methods, this new protocol proved its ability to totally resolve, in a single run, all of the different regioisomeric epoxides. In the long run, this method will demonstrate its efficacy to give insights into the cytochrome P450-dependent metabolism of polyunsatured fatty acids (PUFAs) and the generation of physiologically active epoxy-derivatives.

CYP4A11, the major fatty acid ω‐hydroxylase in human liver is involved in the balance of lipids, but its role and regulation are both poorly understood. We studied the effects of retinoids on the regulation of CYP4A11 in the human hepatoma cell line HepaRG. Treatment of HepaRG cells with all‐trans‐retinoic acid resulted in a strong decrease in CYP4A11 gene expression and apoprotein content and, furthermore, was associated with a 50% decrease in the microsomal lauric acid hydroxylation activity. Such a strong suppression of CYP4A11 expression by retinoids could have a major impact on fatty acid metabolism in the liver.

E-Viniferin, a dimer of resveratrol, was isolated in wine at concentration between 0.5 and 5 muM. As resveratrol and polyphenols from red wine were reported to inhibit cytochrome P450 (CYP) activities, this led us to investigate the inhibitory effects of E-viniferin on human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A activities. These effects were compared to those of resveratrol and non volatiles compounds from red wine or various Cognac(R) beverages (enriched with oak-polyphenols). Assays were carried out on human liver microsomes and heterologously expressed CYPs. Ethoxyresorufin, coumarin, benzoxyresorufin, chlorzoxazone, testosterone and lauric acid were used as selective substrates for CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A, respectively. epsilon-viniferin displayed a more potent inhibitory effect than resveratrol for all the CYP activities tested (Ki 0.5 to 20 muM vs. 10 to 100 muM, respectively). This effect was not due to an inhibition of the NADPH reductase. A particularly potent inhibitory effect was shown for CYP1A1, CYP1B1 and CYP2B6 which are involved in bioactivation of numerous carcinogens. epsilon-viniferin was not a mechanism-based inhibitor of human CYPs. It displayed, like resveratrol, mixed-type inhibitions for all the CYP tested, except for CYP2E1 (non-competitive). Comparison of the inhibitory effects exerted on CYP activities by epsilon-viniferin, resveratrol and non volatile components from red wine or various Cognac beverages showed that neither resveratrol, nor epsilon-viniferin is the main CYP inhibitor present in red wine solids. (C) 2003 Elsevier Science Inc. All rights reserved.

Cytochrome P450 2A6 constitutes 5-10% of the total microsomal CYPs of human liver. Although CYP2A6 is the major coumarin 7-hydroxylase, other known substrates of CYP2A6 include many toxicants and precarcinogens. The chemical structure diversity of these substrates raises the question of their selectivity. Thus, kinetic parameters were determined for the hydroxylation of five substrates of diverse chemical structures known to be selective for cytochrome P450 2A6: methyl tert-butyl ether (MTBE), nicotine, coumarin, N-nitrosobenzylmethylamine (NBzMA), and N-nitrosodiethylamine (NDEA). Sources of enzymes were either human liver microsomes or heterologously expressed CYPs. Coumarin was shown to be the substrate with the highest affinity, followed by NDEA, nicotine, NBzMA, and MTBE. Variability of CYP2A6 catalytic activities in human liver was between 24-fold for MTBE to sevenfold for coumarin, while CYP2A6 content varied 68-fold in human liver microsomes. These five catalytic activities were highly significantly correlated between them and with hepatic CYP2A6 content. The most selective chemical inhibitor of these five substrates was shown to be 8-methoxypsoralen. Based upon chemical inhibition of the enzymatic activities of pure recombinant human CYPs, it cannot be totally excluded that P450s other than CYP2A6, especially CYP2E1, are involved, although to a lesser extent, in NDEA and NBzMA metabolism. In conclusion, the prototype probes for CYP2A6 phenotyping are coumarin and nicotine.