Metabolism Of Eicosapentaenoic Acid Research Articles

Abstract 2182: 15-lipoxygenase-1 modulation of colonic resolvin production to suppress colorectal carcinogenesis

Abstract Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are widely used dietary supplements and FDA-approved treatments. The reported impacts of fish oil, DHA, and EPA on colorectal carcinogenesis (CRC) have spurred debates, as certain studies suggest promotional effects while numerous others emphasize suppressive effects. Nevertheless, resolvins, oxidative metabolites of EPA and DHA, exhibit the ability to inhibit crucial pathways (such as TNF-α and IL-1β) that contribute to CRC. 15-lipoxygenase-1 (ALOX15) plays a crucial role in the oxidative metabolism of DHA and EPA, ultimately leading to the formation of resolvins. However, ALOX15 expression is commonly lost during human CRC. Whether loss of ALOX15 expression modulates the effects of DHA and EPA on CRC remains to be elucidated. We therefore conducted the experiments in preclinical CRC mouse models induced by Azoxymethane or Apc gene mutation to explore the hypothesis that ALOX15 expression in colonic epithelial cells regulates DHA and EPA metabolism to generate resolvins, subsequently influencing CRC outcomes. We found that 1) DHA and EPA exert differential effects on CRC, which can be further modified by factors such as their formulation (e.g., ethyl ester versus triglyceride); 2) targeted transgenic expression of ALOX15 in the intestines of mice (referred to as ALOX15-Gut mice) markedly augmented the production of resolvins and concurrently suppressed CRC; 3) the predominant products from the expressed ALOX15 enzymatic activity were identified as 17-HDHA and resolvin D5; and 4) the effects ALOX15 on increasing resolvin generation and suppressing CRC were consistent across various promoters used to drive ALOX15 transgenic expression in mice. These findings collectively underscore the profound importance of ALOX15 intestinal expression as a host-related factor in resolvin generation from EPA and DHA, which subsequently contributes to the suppression of CRC. Citation Format: Xiangsheng Zuo, Yoshiyuki Kiyasu, Yi Liu, Micheline Moussalli, Bo Wei, Peiying Yang, Imad Shureiqi. 15-lipoxygenase-1 modulation of colonic resolvin production to suppress colorectal carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2182.

Click chemistry-based enrichment strategy for tracing cellular fatty acid metabolism by LC-MS/MS

Fatty acids (FAs), which were initially recognized as energy sources and essential building blocks of biomembranes, serve as the precursors of important signaling molecules. Tracing FA metabolism is essential to understanding the biochemical activity and role of FAs in physiological and pathological events. Inspired by the advances in click chemistry for protein enrichment, we herein established a click chemistry-based enrichment (CCBE) strategy for tracing the cellular metabolism of eicosapentaenoic acid (EPA, 20:5 n-3) in neural cells. Terminal alkyne-labeled EPA (EPAA) used as a surrogate was incubated with N2a, mouse neuroblastoma cells, and alkyne-labeled metabolites (ALMs) were selectively captured by an azide-modified resin via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction for enrichment. After removing unlabeled metabolites, ALMs containing a triazole moiety were cleaved from solid-phase resins and subjected to liquid chromatography mass spectrometry (LC-MS) analysis. The proposed CCBE strategy is highly selective for capturing and enriching alkyne-labeled metabolites from the complicated matrices. In addition, this method can overcome current detection limits by enhancing MS sensitivity of targets, improving the chromatographic separation of sn-position glycerophospholipid regioisomers, facilitating structural characterization of ALMs by a specific MS/MS fragmentation signature, and providing versatile fluorescence detection of ALMs for cellular distribution. This CCBE strategy might be expanded to trace the metabolism of other FAs, small molecules, or drugs.

GWAS-identified bipolar disorder risk allele in the FADS1/2 gene region links mood episodes and unsaturated fatty acid metabolism in mutant mice

Large-scale genome-wide association studies (GWASs) on bipolar disorder (BD) have implicated the involvement of the fatty acid desaturase (FADS) locus. These enzymes (FADS1 and FADS2) are involved in the metabolism of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are thought to potentially benefit patients with mood disorders. To model reductions in the activity of FADS1/2 affected by the susceptibility alleles, we generated mutant mice heterozygously lacking both Fads1/2 genes. We measured wheel-running activity over six months and observed bipolar swings in activity, including hyperactivity and hypoactivity. The hyperactivity episodes, in which activity was far above the norm, usually lasted half a day; mice manifested significantly shorter immobility times on the behavioral despair test performed during these episodes. The hypoactivity episodes, which lasted for several weeks, were accompanied by abnormal circadian rhythms and a marked decrease in wheel running, a spontaneous behavior associated with motivation and reward systems. We comprehensively examined lipid composition in the brain and found that levels of certain lipids were significantly altered between wild-type and the heterozygous mutant mice, but no changes were consistent with both sexes and either DHA or EPA was not altered. However, supplementation with DHA or a mixture of DHA and EPA prevented these episodic behavioral changes. Here we propose that heterozygous Fads1/2 knockout mice are a model of BD with robust constitutive, face, and predictive validity, as administration of the mood stabilizer lithium was also effective. This GWAS-based model helps to clarify how lipids and their metabolisms are involved in the pathogenesis and treatment of BD.

Divergent Metabolic Effects of Metformin Merge to Enhance Eicosapentaenoic Acid Metabolism and Inhibit Ovarian Cancer In Vivo

Simple SummaryAlthough the anticancer effects of metformin are well studied, its effect on energy metabolism of cancer cells remains elusive. Metformin can alter the metabolism of cells either by activating AMPK or inhibiting the mitochondrial respiratory chain complex. The aim of the study was to explore the distinct metabolic profiles of ovarian cancer cell lines post-metformin treatment and to understand metformin’s metabolism-based anti-growth effect on ovarian cancer cell lines. In this study, using the untargeted metabolomics approach, we found that metformin treatment promoted omega-3 fatty acid metabolism in three different ovarian cancer cell lines, and treating ovarian xenografts with specific omega-3 fatty acids, especially EPA, reduced ovarian tumor growth. Thus, our study adds an additional potential therapeutic mechanism to the multi-potent anticancer effects of metformin.Metformin is being actively repurposed for the treatment of gynecologic malignancies including ovarian cancer. We investigated if metformin induces analogous metabolic changes across ovarian cancer cells. Functional metabolic analysis showed metformin caused an immediate and sustained decrease in oxygen consumption while increasing glycolysis across A2780, C200, and SKOV3ip cell lines. Untargeted metabolomics showed metformin to have differential effects on glycolysis and TCA cycle metabolites, while consistent increased fatty acid oxidation intermediates were observed across the three cell lines. Metabolite set enrichment analysis showed alpha-linolenic/linoleic acid metabolism as being most upregulated. Downstream mediators of the alpha-linolenic/linoleic acid metabolism, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were abundant in all three cell lines. EPA was more effective in inhibiting SKOV3 and CaOV3 xenografts, which correlated with inhibition of inflammatory markers and indicated a role for EPA-derived specialized pro-resolving mediators such as Resolvin E1. Thus, modulation of the metabolism of omega-3 fatty acids and their anti-inflammatory signaling molecules appears to be one of the common mechanisms of metformin’s antitumor activity. The distinct metabolic signature of the tumors may indicate metformin response and aid the preclinical and clinical interpretation of metformin therapy in ovarian and other cancers.

A structurally engineered fatty acid, icosabutate, suppresses liver inflammation and fibrosis in NASH

Although long-chain omega-3 fatty acids (LCn-3FAs) regulate inflammatory pathways of relevance to non-alcoholic steatohepatitis (NASH), their susceptibility to peroxidation may limit their therapeutic potential. We compared the metabolism of eicosapentaenoic acid (EPA) with an engineered EPA derivative (icosabutate) in human hepatocytes invitro and their effects on hepatic glutathione metabolism, oxidised lipids, inflammation, and fibrosis in a dietary mouse model of NASH, and in patients prone to fatty liver disease. Oxidation rates and cellular partitioning of EPA and icosabutate were compared in primary human hepatocytes. Comparative effects of delayed treatment with either low- (56mg/kg) or high-dose (112mg/kg) icosabutate were compared with EPA (91mg/kg) or a glucagon-like peptide 1 receptor agonist in a choline-deficient (CD), L-amino acid-defined NASH mouse model. To assess the translational potential of these findings, effects on elevated liver enzymes and fibrosis-4 (FIB-4) score were assessed in overweight, hyperlipidaemic patients at an increased risk of NASH. In contrast to EPA, icosabutate resisted oxidation and incorporation into hepatocytes. Icosabutate also reduced inflammation and fibrosis in conjunction with a reversal of CD diet-induced changes in the hepatic lipidome. EPA had minimal effect on any parameter and even worsened fibrosis in association with depletion of hepatic glutathione. In dyslipidaemic patients at risk of NASH, icosabutate rapidly normalised elevated plasma ALT, GGT and AST and reduced FIB-4 in patients with elevated ALT and/or AST. Icosabutate does not accumulate in hepatocytes and confers beneficial effects on hepatic oxidative stress, inflammation and fibrosis in mice. In conjunction with reductions in markers of liver injury in hyperlipidaemic patients, these findings suggest that structural engineering of LCn-3FAs offers a novel approach for the treatment of NASH. Long-chain omega-3 fatty acids are involved in multiple pathways regulating hepatic inflammation and fibrosis, but their susceptibility to peroxidation and use as an energy source may limit their clinical efficacy. Herein, we show that a structurally modified omega-3 fatty acid, icosabutate, overcame these challenges and had markedly improved antifibrotic efficacy in a mouse model of non-alcoholic steatohepatitis. A hepatoprotective effect of icosabutate was also observed in patients with elevated circulating lipids, in whom it led to rapid reductions in markers of liver injury.

The functional loss of ceramide metabolism enzymes ceramide synthase and acid sphingomyelinase on lipid metabolism and healthspan

The increase in our aging population in combination with the growing incidence of late-life conditions presents a need for research on healthy aging. Here, we present work examining the roles of sphingolipid metabolism on aging. Sphingolipids play important roles in stress response, cell survival, cell signaling, and aging. Specifically, ceramide responds to oxidative stress and recruits apoptotic proteins to the cell membrane thereby causing cell cycle arrest. Furthermore, ceramide levels increase with age, making it an important lipid mediator of aging. The objective of this study is to examine the physiological role of ceramide metabolism on aging and lipid metabolism pathways in the roundworm, Caenorhabditis elegans. To assess these mechanisms, we utilized C. elegans’ mutants lacking acid sphingomyelinase (asm-3) and ceramide synthase (hyl-2) which we previously found to be long- and short-lived respectively. We performed a lipidomic analysis to explore longitudinal changes in lipid concentrations in wild type (N2), asm-3/acid sphingomyelinase, and hyl-2/ceramide synthase worms at 1, 5, and 10 days of age. Among our samples, we detected 700 different lipids that were abundant enough in concentration for analysis. Interestingly, 10 day old hyl-2 mutants, which have a reduced life-span, showed an increased concentration of eicosapentaenoic acid (EPA), an omega-3 fatty acid that has been shown to increase with longevity in worms. Conversely, asm-3 mutants, which are long-lived animals, have reduced levels of EPA. To expand upon our lipidomic data with enzymatic findings, we utilized RT-qPCR analysis to longitudinally analyze fatty acid desaturases (fat-1 and fat-4) involved in EPA metabolism in worms. We found that fat-4 expression is reduced in 1, 5, and 10 day hyl-2 animals, suggesting that ceramide metabolism may impact fatty acid genes involved in aging. Future work will examine the role of stress response genes including superoxide dismutase (sod-3) and fatty acid tail elongases (elo-5 and elo-6) in 1, 5, and 10 day old hyl-2 and asm-3 mutants. Finally, to expand upon the current knowledge of ceramide's role in lifespan, we present data on the RNAi mediated suppression of hyl-2 in long-lived mutants including mitochondrial respiration (clk-1/coenzyme Q), insulin-like signaling (daf-2/insulin-like receptor), and dietary starvation (eat-2/mAChR). Our goal is to identify whether ceramide metabolism works within these known pathways that mediate aging. With these assays, we hope to better understand the intrinsic biochemical lipid processes associated with ceramide metabolism in aging animals.

Different metabolism of EPA, DPA and DHA in humans: A double-blind cross-over study

This study aimed to compare eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) incorporated into red blood cells (RBC) phospholipids (PL), plasma PL, plasma triglyceride (TAG), and plasma cholesteryl ester (CE) fractions, and the metabolomics profiles in a double-blind cross-over study. Twelve female healthy subjects randomly consumed 1 g per day for 6 days of pure EPA, DPA, or DHA. The placebo treatment was olive oil. The fasting venous blood was taken at days 0, 3 and 6, and the RBC PL and plasma lipid fractions were separated for fatty acid determination using thin layer chromatography followed by gas chromatography. Plasma metabolites were analyzed by UHPLC-Q-Exactive Orbitrap/MS. Supplemental EPA significantly increased the concentrations of EPA in RBC PL (days 3 and 6). For subjects consuming the DPA supplement, the concentrations of both DPA and EPA were significantly increased in RBC PL over a 6-day period, respectively. For plasma PL fraction, EPA and DPA supplementation significantly increased the concentrations of EPA and DPA at both days 3 and 6, respectively. Supplemental DHA significantly increased the concentrations of DHA in plasma PL at day 6. For plasma TAG fraction, supplementation with EPA and DPA significantly increased the concentrations of EPA and DPA at both days 3 and 6, respectively. After DHA supplementation, significant increases in the concentrations of DHA were found relative to baseline at both days 3 and 6. For plasma CE fraction, EPA supplementation significantly increased the concentrations of EPA (days 3 and 6) and DPA (days 6), respectively. Supplemental DPA significantly increased the concentrations of EPA at day 6. Meanwhile, the concentrations of DHA were significantly increased over a 6-day period of intervention after subjects consuming the DHA supplements. There were a total of 922 plasma metabolites identified using metabolomics analyses. Supplementation with DPA and DHA significantly increased the levels of sphingosine 1-phosphate (P for DPA = 0.025, P for DHA = 0.029) and 15-deoxy-Δ12,14-prostaglandin A1 (P for DPA = 0.034; P for DHA = 0.021) in comparison with olive oil group. Additionally, supplementation with EPA (P = 0.007) and DHA (P = 0.005) significantly reduced the levels of linoleyl carnitine, compared with olive oil group. This study shows that DPA might act as a reservoir of n-3 LCP incorporated into blood lipid fractions, metabolized into DHA, and retro-converted back to EPA. Metabolomics analyses indicate that supplemental EPA, DPA and DHA have shared and differentiated metabolites. The differences of these metabolic biomarkers should be investigated in additional studies.

The molecular basis of essential fatty acid limitation in Daphnia magna: A transcriptomic approach.

It is widely accepted that in many food webs, the trophic transfer efficiency among primary producers and herbivores is determined by the nutritional value of primary producers. In pelagic freshwater and marine ecosystems, secondary production by herbivorous crustacean zooplankton is often limited by the seston's content of essential ω3 polyunsaturated fatty acids (ω3 PUFAs). However, little is known about the genetic network behind the positive relationship between phytoplankton ω3 PUFA content and zooplankton growth and reproduction. In our experimental study, we analysed gene expression changes of the freshwater cladoceran Daphnia magna under different food regimes differing in their ω3 PUFA composition. To disentangle ω3 PUFA effects from other factors, we fed D.magna with different pure phytoplankton cultures (i.e., algal and cyanobacterial diets) with or without supplementing the essential ω3 PUFA eicosapentaenoic acid (EPA). As hypothesized, we observed enhanced growth on diets supplemented with EPA. We applied an Illumina RNA-seq approach to D.magna from different diet treatments to find and monitor genes that are regulated dependent on EPA availability. Of 26,646 potential protein products (mapped to the D.magna genome), we identified transcriptomic signatures driven by the different food sources. Further analyses revealed specific candidate genes involved in EPA metabolism, irrespective of the basal food source. This allows a first functional annotation of previously uncharacterized genes involved in the EPA-specific response of D.magna and may finally provide a link to molecular processes connected to ω3 PUFA metabolism and conversion and thus trophic transfer efficiency in pelagic food webs.

Arachidonic Acid and Eicosapentaenoic Acid Metabolism in Juvenile Atlantic Salmon as Affected by Water Temperature.

Salmons raised in aquaculture farms around the world are increasingly subjected to sub-optimal environmental conditions, such as high water temperatures during summer seasons. Aerobic scope increases and lipid metabolism changes are known plasticity responses of fish for a better acclimation to high water temperature. The present study aimed at investigating the effect of high water temperature on the regulation of fatty acid metabolism in juvenile Atlantic salmon fed different dietary ARA/EPA ratios (arachidonic acid, 20:4n-6/ eicosapentaenoic acid, 20:5n-3), with particular focus on apparent in vivo enzyme activities and gene expression of lipid metabolism pathways. Three experimental diets were formulated to be identical, except for the ratio EPA/ARA, and fed to triplicate groups of Atlantic salmon (Salmo salar) kept either at 10°C or 20°C. Results showed that fatty acid metabolic utilisation, and likely also their dietary requirements for optimal performance, can be affected by changes in their relative levels and by environmental temperature in Atlantic salmon. Thus, the increase in temperature, independently from dietary treatment, had a significant effect on the β-oxidation of a fatty acid including EPA, as observed by the apparent in vivo enzyme activity and mRNA expression of pparα -transcription factor in lipid metabolism, including β-oxidation genes- and cpt1 -key enzyme responsible for the movement of LC-PUFA from the cytosol into the mitochondria for β-oxidation-, were both increased at the higher water temperature. An interesting interaction was observed in the transcription and in vivo enzyme activity of Δ5fad–time-limiting enzyme in the biosynthesis pathway of EPA and ARA. Such, at lower temperature, the highest mRNA expression and enzyme activity was recorded in fish with limited supply of dietary EPA, whereas at higher temperature these were recorded in fish with limited ARA supply. In consideration that fish at higher water temperature recorded a significantly increased feed intake, these results clearly suggested that at high, sub-optimal water temperature, fish metabolism attempted to increment its overall ARA status -the most bioactive LC-PUFA participating in the inflammatory response- by modulating the metabolic fate of dietary ARA (expressed as % of net intake), reducing its β-oxidation and favouring synthesis and deposition. This correlates also with results from other recent studies showing that both immune- and stress- responses in fish are up regulated in fish held at high temperatures. This is a novel and fundamental information that warrants industry and scientific attention, in consideration of the imminent increase in water temperatures, continuous expansion of aquaculture operations, resources utilisation in aquafeed and much needed seasonal/adaptive nutritional strategies.

Specificity of Lipoxygenase Pathways Supports Species Delineation in the Marine Diatom Genus Pseudo-nitzschia

Oxylipins are low-molecular weight secondary metabolites derived from the incorporation of oxygen into the carbon chains of polyunsaturated fatty acids (PUFAs). Oxylipins are produced in many prokaryotic and eukaryotic lineages where they are involved in a broad spectrum of actions spanning from stress and defense responses, regulation of growth and development, signaling, and innate immunity. We explored the diversity in oxylipin patterns in the marine planktonic diatom Pseudo-nitzschia. This genus includes several species only distinguishable with the aid of molecular markers. Oxylipin profiles of cultured strains were obtained by reverse phase column on a liquid chromatograph equipped with UV photodiode detector and q-ToF mass spectrometer. Lipoxygenase compounds were mapped on phylogenies of the genus Pseudo-nitzschia inferred from the nuclear encoded hyper-variable region of the LSU rDNA and the plastid encoded rbcL. Results showed that the genus Pseudo-nitzschia exhibits a rich and varied lipoxygenase metabolism of eicosapentaenoic acid (EPA), with a high level of specificity for oxylipin markers that generally corroborated the genotypic delineation, even among genetically closely related cryptic species. These results suggest that oxylipin profiles constitute additional identification tools for Pseudo-nitzschia species providing a functional support to species delineation obtained with molecular markers and morphological traits. The exploration of the diversity, patterns and plasticity of oxylipin production across diatom species and genera will also provide insights on the ecological functions of these secondary metabolites and on the selective pressures driving their diversification.

Naturally occurring monoepoxides of eicosapentaenoic acid and docosahexaenoic acid are bioactive antihyperalgesic lipids

Beneficial physiological effects of long-chain n-3 polyunsaturated fatty acids are widely accepted but the mechanism(s) by which these fatty acids act remains unclear. Herein, we report the presence, distribution, and regulation of the levels of n-3 epoxy-fatty acids by soluble epoxide hydrolase (sEH) and a direct antinociceptive role of n-3 epoxy-fatty acids, specifically those originating from docosahexaenoic acid (DHA). The monoepoxides of the C18:1 to C22:6 fatty acids in both the n-6 and n-3 series were prepared and the individual regioisomers purified. The kinetic constants of the hydrolysis of the pure regioisomers by sEH were measured. Surprisingly, the best substrates are the mid-chain DHA epoxides. We also demonstrate that the DHA epoxides are present in considerable amounts in the rat central nervous system. Furthermore, using an animal model of pain associated with inflammation, we show that DHA epoxides, but neither the parent fatty acid nor the corresponding diols, selectively modulate nociceptive pathophysiology. Our findings support an important function of epoxy-fatty acids in the n-3 series in modulating nociceptive signaling. Consequently, the DHA and eicosapentaenoic acid epoxides may be responsible for some of the beneficial effects associated with dietary n-3 fatty acid intake.

Stereoselective epoxidation of the last double bond of polyunsaturated fatty acids by human cytochromes P450

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.

Abstract 4129: Effects of the metabolism of omega-3 fatty acid EPA by CYP1A1 in breast cancer proliferation and survival

Abstract Omega-3 fatty acids are widely known for their importance in heart disease. However, the impact of omega-3 fatty acids in breast cancer is not fully understood. Through these studies we seek to understand the role of the metabolism of the omega-3 fatty acid known as eicosapentaenoic acid (EPA) in breast cancer and determine whether it may affect breast cancer progression. It is known that high levels (60uM) of EPA have anti-proliferative effects in various cancer lines. Consistent with these reports, we found that EPA treatment (60uM) of the estrogen receptor positive line MCF7 and the estrogen receptor negative line MDA-MB-231 decreases cell proliferation. Interestingly, lower dosing of EPA (15uM-40uM) significantly stimulated the proliferation of these lines. It has been shown that cytochrome P450 1A1 (CYP1A1) selectively epoxygenates EPA to 17(18)-epoxyeicosatetraenoic acid (17,18-EpETE). Measurement of cell growth by MTT assay shows that 17,18-EpETE promotes the proliferation of the MCF7 and MDA-MB-231 lines in a dose- (≥ 1uM) and time-dependent (max effect at 48-72hrs) manner. Together, these results led us to hypothesize that CYP1A1 metabolizes EPA into 17,18-EpETE, thereby promoting breast cancer proliferation and survival. To better understand this epoxygenase mechanism, we studied the impact of CYP1A1 and the 17,18-EpETE metabolite of EPA on cell proliferation by MTT assay, cell cycle progression by propidium iodine staining, and apoptosis by Annexin V flow cytometry. We found that CYP1A1 is expressed in MCF7 and MDA-MB-231 lines and siRNA-mediated knock down significantly inhibits the proliferation of these lines. CYP1A1 knock down also blocks the proliferative effect of treatment with 15uM EPA. Our preliminary data also indicate that 17,18-EpETE promotes growth of the MDA-MB-231 line, in part, by decreasing apoptosis and increasing the S- and G2/M cell populations. Consistently, knock down of CYP1A1 in MDA-MB-231 line increases the G0/1 cell population and decreases number of cells in the S and G2/M phases, consistent with a G1 block. The mechanism of action in MCF7 cells is yet to be determined. Better understanding of the mechanism by which EPA metabolism affects breast cancer may impact our understanding of breast cancer prevention and therapeutics. We propose that CYP1A1 may be of significant importance in breast cancer progression through novel mechanisms that depend on biosynthetic pathways promoting EPA epoxygenation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4129.

A combination study of spin-trapping, LC/ESR and LC/MS on carbon-centred radicals formed from lipoxygenase-catalysed peroxidation of eicosapentaenoic acid

Increased evidence from animal and in vitro cellular research indicates that the metabolism of eicosapentaenoic acid (EPA) can inhibit carcinogenesis in many cancers. Free radical-mediated peroxidation is one of many possible mechanisms to which EPA's anti-cancer activity has been attributed. However, no direct evidence has been obtained for the formation of any EPA-derived radicals. In this study, a combination of LC/ESR and LC/MS was used with α-[4-pyridyl 1-oxide]-N-tert-butyl nitrone to identify the carbon-centred radicals that are formed in lipoxygenase-catalysed EPA peroxidation. Of the numerous EPA-derived radicals observed, the major products were those stemming from β-scission of 5-, 15- and 18-EPA-alkoxyl radicals. By means of an internal standard in LC/MS, this study also quantified each radical adduct in all its redox forms, including an ESR-active form and two ESR-silent forms. The comprehensive profile of EPA's radical formation provides a starting point for ongoing research in defining the biological effects of radicals generated from EPA peroxidation.

Cytochrome P450-dependent metabolism of eicosapentaenoic acid in the nematode Caenorhabditis elegans

The genome of Caenorhabditis elegans contains 75 full length cytochrome P450 (CYP) genes whose individual functions are largely unknown yet. We tested the hypothesis that some of them may be involved in the metabolism of eicosapentaenoic acid (EPA), the predominant polyunsaturated fatty acid of this nematode. Microsomes isolated from adult worms contained spectrally active CYP proteins and showed NADPH-CYP reductase (CPR) activities. They metabolized EPA and with lower activity also arachidonic acid (AA) to specific sets of regioisomeric epoxy- and ω-/(ω-1)-hydroxy-derivatives. 17( R),18( S)-epoxyeicosatetraenoic acid was produced as the main EPA metabolite with an enantiomeric purity of 72%. The epoxygenase and hydroxylase reactions were NADPH-dependent, required the functional expression of the CPR-encoding emb-8 gene, and were inhibited by 17-ODYA and PPOH, two compounds known to inactivate mammalian AA-metabolizing CYP isoforms. Multiple followed by single RNAi gene silencing experiments identified CYP-29A3 and CYP-33E2 as the major isoforms contributing to EPA metabolism in C. elegans. Liquid chromatography/mass spectrometry revealed that regioisomeric epoxy- and hydroxy-derivatives of EPA and AA are endogenous constituents of C. elegans. The endogenous EPA metabolite levels were increased by treating the worms with fenofibrate, which also induced the microsomal epoxygenase and hydroxylase activities. These results demonstrate for the first time that C. elegans shares with mammals the capacity to produce CYP-dependent eicosanoids and may thus facilitate future studies on the mechanisms of action of this important class of signaling molecules.

Probing the skin permeation of eicosapentaenoic acid and ketoprofen: 2. Comparative depth profiling and metabolism of eicosapentaenoic acid

Unexpected enhancement of the topical delivery of eicosapentaenoic acid (EPA) across porcine skin was observed previously when fish oil was co-formulated with ketoprofen. In the current work depth profile analysis was used to probe the epidermal conversion of EPA to its 15-hydroxy metabolite in the presence and absence of ketoprofen. Freshly excised full-thickness porcine skin in Franz diffusion cells was dosed (both infinite and finite) with simple formulations based on fish oil as source of EPA. After 24 h the skin was subjected to tape stripping and depth profiles were constructed. Typical depth profiles were obtained, with an inverse relationship between depth and permeant concentration. 15-HEPE was generated in the skin when Hepes-modified Hanks’ balanced salt solution was used, but none was detected when a cetrimide receptor phase was used, highlighting the importance of maintaining skin viability in such exercises. Ketoprofen had a direct influence on the metabolism of EPA and resulting in conversion to its 15-LOX metabolite 15-HEPE. However, this link appears to be only part of the solution of EPA enhancement however, as even in non-viable skin ketoprofen had an enhancing affect.

Human CYP1A1 variants lead to differential eicosapentaenoic acid metabolite patterns

To answer the question whether the most common allelic variants of human CYP1A1, namely CYP1A1.1 (wild type), CYP1A1.2 (Ile462Val), and CYP1A1.4 (Thr461Asn), differ in their catalytic activity towards eicosapentaenoic acid (EPA), in vitro enzymatic assays were performed in reconstituted CYP1A1 systems. All CYP1A1 variants catalyzed EPA epoxygenation and hydroxylation to 17( R),18( S)-epoxyeicosatetraenoic acid (17( R),18( S)-EETeTr) and 19-OH-EPA, yet with varying catalytic efficiency and distinct regiospecificity. CYP1A1.1 and CYP1A1.4 formed 17( R),18( S)-EETeTr as main product ( K m = 53 and 50 μM; V max = 0.60 and 0.50 pmol/min/pmol; V max/ K m = 0.11 and 0.10 μM −1 min −1, respectively), followed by 19-OH-EPA ( K m = 76 and 93 μM; V max = 0.37 and 0.37 pmol/min/pmol; V max/ K m = 0.005 and 0.004 μM −1 min −1, respectively). The variant CYP1A1.2 produced almost equal amounts of both metabolites, but its catalytic efficiency for hydroxylation was five times higher ( K m = 66 μM; V max = 1.7 pmol/min/pmol; V max/ K m = 0.026 μM −1 min −1) and that for epoxygenation was twice higher ( K m = 66 μM; V max = 1.5 pmol/min/pmol; V max/ K m = 0.023 μM −1 min −1) than those of the wild-type enzyme. Thus, the Ile462Val polymorphism in human CYP1A1 affects EPA metabolism and may contribute to interindividual variance in the local production of physiologically active fatty acid metabolites in the cardiovascular system and other extrahepatic tissues, where CYP1A1 is expressed or induced by polycyclic aromatic hydrocarbons and other xenobiotics.

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.

Arachidonic and eicosapentaenoic acid metabolism by human CYP1A1: highly stereoselective formation of 17( R),18( S)-epoxyeicosatetraenoic acid

Human cytochrome P450 1A1 (CYP1A1) and human NADPH-cytochrome P450 reductase were expressed and purified from Spodoptera frugiperda insect cells. A reconstituted enzymatically active system metabolized polyunsaturated fatty acids such as arachidonic (AA) and eicosapentaenoic acid (EPA). CYP1A1 was an AA hydroxylase which oxidizes this substrate at a rate of 650±10 pmol/min/nmol CYP1A1, with over 90% of metabolites accounted for by hydroxylation products and with 19-OH-AA as major product. Epoxyeicosatrienoic acid (EET), mainly 14,15-EET, accounted for about 7% of total metabolites. Unlike rat CYP1A1, the human enzyme exhibited no 20-OH-AA as product. In contrast, with EPA as substrate CYP1A1 was mainly an epoxygenase, oxidizing with over 68% of total metabolites EPA to 17( R),18( S)-epoxyeicosatetraenoic acid (17( R),18( S)-EETeTr). 19-OH-EPA accounted for about 31% of total metabolites. Significantly, the 17,18-olefinic bond of EPA was epoxidized to 17( R),18( S)-EETeTr with nearly absolute regio- and stereoselectivity. Molecular modeling analyses provided rationale for high efficiency of AA hydroxylation at C 19 and its gradual decrease down to C 14, as well as for the limited EPA 17( S),18( R) epoxidation due to unfavorable enzyme–substrate interactions. The absence of ω-hydroxylation for both substrates is not due to steric factors, but probably a consequence of different reactivities of ω and (ω−1) carbons for hydrogen abstraction. It is suggested that the capacity of human CYP1A1 to metabolize AA and EPA and its inducibility by polycyclic aromatic hydrocarbons may affect the production of physiologically active metabolites, in particular, in the cardiovascular system and other extrahepatic tissues including lung.

Metabolism of eicosapentaenoic acid by CYP4A and CYP2C enzymes

Metabolism of eicosapentaenoic acid by CYP4A and CYP2C enzymes