Littérature scientifique sur le sujet « CYP450-derived eicosanoids »

Abstract Arachidonic acid (AA) metabolizing enzymes, including cyclooxygenases (COX), lipoxygenases, and cytochrome P450s (CYP450), are expressed in breast cancer (BC). Elevated levels of metabolites of COX-derived, pro-inflammatory prostaglandin (PGs) metabolites in urine have been linked to increased risk of developing breast cancer (BC) in two large prospective cohorts. Further, CYP450-derived EETs in BC tumors are associated with metastasis. We hypothesize that measuring levels of AA-derived oxylipins in breast tissues could enable improved BC risk prediction, provide insights into pro-carcinogenic inflammatory mechanisms, and serve as potential surrogate endpoints in chemoprevention trials. Thus, we performed a pilot study to assess the feasibility of measuring these analytes in small breast tissue samples. We analyzed two sample sets, collected under Institutional Review Board (IRB) approved protocols: 50 normal breast tissue cores donated to the Komen Tissue Bank (KTB) via vacuum-assisted biopsy and 10 tissues removed surgically at Mayo Clinic beyond resection margins of lumpectomy samples performed for atypical ductal hyperplasia (ADH). Tissues were frozen and maintained at -80°C. Normal KTB tissue cores were split in half. Tissues for analysis were weighed, extracted in cold methanol containing a cyclooxygenase inhibitor, and assayed for a panel of 42 oxylipins using validated mass spectrometric methods with a limit of detection per analyte of 0.010 ng/mL in supernatant and CVs of approximately <11%. Analyte measurements in paired cores (ng/g tissue) were compared with Spearman correlations and intra-class correlation coefficients. Measurements in surgical samples were assessed descriptively and levels were compared with normal KTB breast tissues.Median weight of the 50 split KTB cores was 0.055 grams. 17 oxylipins were detected in the tissue samples. PGD2 demonstrated the weakest correlation between split cores (r=0.12; p=0.40), whereas 15-HETE demonstrated the strongest correlation (r=0.68; p<0.0001). ICCs ranged from non-significant to nearly perfect, with highest values for 12-HETE (ICC=0.98, 95%CI: 0.97-0.99). Exploratory analyses of the KTB cores demonstrated significant associations between two BC risk factors, alcohol use and family history of breast/ovarian cancer, and several analytes. Levels of most eicosanoids tested were substantially and highly significantly elevated in tissues surrounding ADH versus KTB normal breast tissues. We conclude that eicosanoids can be readily measured in small frozen breast tissue samples obtained via percutaneous biopsy or at surgery. Future studies to assess the associations of eicosanoid levels with BC risk factors and development of BC after a benign biopsy may inform risk prediction and potentially inhibitable inflammatory mechanisms of breast carcinogenesis. Citation Format: Ginger L. Milne, Robert A. Vierkant, Amy C. Degnim, Anna Maria Storniolo, Jill E. Henry, Laura M. Pacheco-Spann, Derek C. Radisky, Mark E. Sherman. Quantification of oxylipins as biomarkers of inflammation and cancer risk in breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6333.

BackgroundArachidonic acid (AA) is a polyunsaturated fatty acid, released in inflammatory disease, such as rheumatoid arthritis (RA). Cyclooxygenase (COX) and lypooxygenase (LOX) pathways have received greater attention than cytochrome P450 (CYP) pathway of AA, which also plays a significant role in RA. AA is a substrate of CYP enzymes through two different pathways: the ω-hydroxylase, and epoxygenase pathways, respectively. The epoxygenase gives rise to epoxyeicosatrienoic acids (EETs) (5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET), whereas ω- hydroxylase produces hydroxyeicosatetraenoic acids (HETEs) [1].ObjectivesThe aim of this study is to evaluate the role of Cytochrome P450 metabolites of arachidonic acid and their therapeutic targeting in rheumatoid arthritis.MethodsPubmed and Scopus databases were the main databases used to identify all the studies conformed to the eligibility criteria.ResultsCYP enzymes (CYP3A, CYP2C19, CYP2C9, CYP1A2) suppression was observed in the synovial fluid of RA patients with higher IL-6 levels. EETs inhibit bone resorption and osteoclastogenesis, have an important role in blocking inflammation by reducing TNFα, and are negatively linked with pro-inflammatory cytokines (IL-1, IL-6, IL-8)[2]. The anti-inflammatory effects of EETs are potentially attributed to peroxisome proliferator–activated receptor gamma (PPARγ) activation. EETs are also metabolized through soluble epoxide hydrolase (sEH) to dihydroxyeicsatrienoic acids (DHETs), which have a pro-inflammatory activity. On the other hand, 20-HETE deriving from CYP ω-hydroxylase pathway has pro-inflammatory effect [3]. Data have shown that the genetic variations of CYP could affect the individual susceptibility to RA [4].ConclusionsEH inhibitors (sEHi) can not only block the inflammation caused by EETs metabolite, but can also act on LOX and COX pathway, and can be used in chronic-phase arthritis to reduce both inflammation, and the pain. In a mouse model of RA, sEHi showed decent RA assessment score improvement [5]. Moreover, data suggest that sEHi inhibit also PGE2 production, and exert an additional anti-inflammatory effect in arthritis. Since NSAIDs can lead to gastric and cardiovascular problems, sEHi are considered a better pharmacological approach in inflammatory rheumatic disease. Despite the failure of some of sEHi to reach clinical trials, we believe that CYP-derived eicosanoids should be further studied as potential target in rheumatic disease. Dual inhibitors sEHI/5-LOX activating protein (FLAP) are also very promising compounds as they can inhibit leukotriene formation, without effecting the levels of anti-inflammatory pro-resolving mediators. In perspective we suggest that multiple ligands targeting different AA pathways or mediators should be further explored as potential targets for designing new compounds to treat RA patients.References[1]M. Hoxha, B. Zappacosta. CYP-derived eicosanoids: Implications for rheumatoid arthritis. Prostaglandins Other Lipid Mediat. 146 (2020) 106405.[2]X. Xu, X.A. Zhang, D.W. Wang, The roles of CYP450 epoxygenases and metabolites, epoxyeicosatrienoic acids, in cardiovascular and malignant diseases, Adv. Drug Deliv. Rev. 63 (8) (2011) 597–609.[3]D. Panigrahy, A. Kaipainen, E.R. Greene, S. Huang, Cytochrome P450-derived eicosanoids: the neglected pathway in cancer, Cancer Metastasis Rev. 29 (4) (2010) 723–735[4]S. Puntarulo, A.I. Cederbaum, Production of reactive oxygen species by microsomes enriched in specific human cytochrome P450 enzymes, Free Radic. Biol. Med. 24 (7–8) (1998) 1324–1330[5]J. Park, M.J. Cho, H.J. Park, Analgesic effects of soluble epoxide hydrolase inhibitor in K/BxN serum transfer arthritis mouse model, Anesth. Pain Med. 14 (2019) 76–94.Disclosure of InterestsNone declared

Background:traditional approaches and lipidomics support the relevance of eicosanoids in rheumatic conditions, such as rheumatoid arthritis (RA). Previous studies from our group and others have revealed altered levels of arachidonic acid in RA, pointing to a possible consumption or enhanced metabolisation. However, the potential alterations of its actual metabolites are difficult to assess with conventional approaches, and new, untargeted, high-throughput technologies are needed. More importantly, whether these alterations are related to the disease course and could be found at the very early stage of the disease is unknown.Objectives:to characterize the eicosanoid profiles during the earliest stages of RA and their potential clinical associations.Methods:60 very early RA patients (50 recruited at diagnosis and treatment-naïve) fulfilling 2010 ACR/EULAR criteria, 11 clinical suspect arthralgia (CSA) individuals and 28 healthy controls (HC) were recruited. Samples were collected at the moment of the diagnosis. Serum oxylipins profiles were analyzed by mass spectrometry (LC-MS/MS). Treatment-naïve patients underwent csDMARD treatment and were followed for 6 (n=49) and 12 months (n=38). Data analysis was performed in R and MetaboAnalyst.Results:A total of 75 oxylipins, mostly derived from arachidonic (AA), eicosapentanoic (EPA) and linoleic (LA) acid, were identified. No effect was observed for age, gender or BMI. Correlation and network analyses revealed different patterns among oxylipins across RA patients, CSA and HC (Figure 1A). The 8-HETrE, PGE3 and 20-HETE showed the pattern (linear increase) HC→CSA→RA (p=1.47·10-4, 5.34·10-4 and 5.68·10-4, respectively; and adjusted FDR<0.050) (Figure 1B). A PLS-DA (explaining 12.3% of the total variance, with a 71.0% cross-validation accuracy and permutation p=5·10-4) confirmed that oxylipins profiles differ among groups, although a certain overlap existed. A total of 22 oxylipins had VIP scores>1 (Figure 1C), which allowed the identification of two clusters (I and II). Cluster usage (I/II) differed among groups (p=0.003): HC (27/1), CSA (7/4) and RA (37/23). Patients exhibiting cluster II showed higher VAS global assessment (p=0.016) and pain (p=0.003) than their cluster I-counterparts. More importantly, cluster II patients were less likely to achieve DAS28 remission at 6 (12/17 vs 10/32, p=0.008) and 12 months (6/9 vs 9/29, p=0.066) upon conventional DMARD treatment compared to those showing cluster I.OPLS-DA analyses revealed a good discrimination between CSA and HC groups, and 7 compounds (13-HODE, PGB2, 9-oxo-ODE, 12-oxo-ETE, 19,20-di-HDPA, 5-HETrE and 15-HEPE) were associated with the course HC->CSA. Different precursors (2 LA, 3 AAA, 1 EPA and 1 DHA) and pathways were noted (3 LOX, 4 CYP450). Regarding RA subsets, differences were noted by seropositivity. Whereas 9 compounds were associated with the pattern HC→seronegative RA (8-HETrE, PGE3, 20-HETE, 19,20-di-HDPA, PGEM, PGJ2, 12-oxo-LTB4, 14,15-EET and LTB4), a distinct set was observed for the pattern HC→seropositive RA (PGE3, 20-oh-PGE2, 5-HETE, PGE2, 12-oxo-ETE, 20-HETE, PGEM, 4-HDoHE, LTB4, 9-oxo-ODE, 12-oxo-LTB4, 8,9-EET). No differences in the major pathways were noted.Conclusion:Oxylipin networks differ across disease stages during the very early phase of RA, and can inform on specific signatures related to the disease progression. Oxylipins can delineate profiles with clinical relevance and are able to predict treatment response.Figure:Disclosure of Interests:None declared

We evaluated the contribution of cytochrome P-450 (CYP450)-dependent arachidonic acid (AA) metabolites and prostanoids to the renal hemodynamic and tubular effects of endothelin-1 (ET-1) in anesthetized rats. Either ET-1 (0.3, 1.0, and 3 pmol ⋅ kg−1 ⋅ min−1) or vehicle was infused intravenously during two to three 30-min clearance experimental periods. Only high-dose ET-1 increased mean arterial pressure: control, 75 ± 3 mmHg vs. experimental, 84 ± 4 mmHg. A dose-dependent diuretic-natriuretic response to ET-1 occurred despite progressive declines in glomerular filtration rate (GFR) and renal blood flow. In the face of a 36% reduction in GFR in response to the highest dose of ET-1, urinary sodium excretion (UNaV) increased threefold from 0.57 ± 0.11 to 1.6 ± 0.10 μmol ⋅ 100 g−1 ⋅ min−1. Indomethacin (5 mg/kg) decreased basal GFR from 1.2 ± 0.3 ml ⋅ 100 g−1 ⋅ min−1to 0.8 ± 0.1 ml ⋅ 100 g−1 ⋅ min−1and potentiated the GFR lowering action of ET-1 associated with reductions in UNaV and urine volume. Cobalt chloride (CoCl2) and dibromododec-11-enoic acid (DBDD), which diminish CYP450-dependent AA metabolism through different mechanisms, were used to identify CYP450 products mediating the renal functional actions of ET-1. DBDD (12.5 μg/min) reduced urinary excretion of 20-hydroxyeicosatetraenoic acid from 3.4 ± 0.9 (control) to 1.1 ± 0.6 ng/h and abolished the negative effects of ET-1 on GFR while decreasing the diuretic-natriuretic action of ET-1. Similar effects were produced by CoCl2. Clotrimazole, an inhibitor of epoxygenase activity, was without effect on ET-1-induced renal functional changes. Thus the capacity of ET-1 to enhance prostaglandin production was primarily expressed in terms of positive effects on renal hemodynamics. In contrast, CYP450 products promoted sodium excretion despite negative effects on renal hemodynamics.

Endothelin-1 (ET-1) produces potent renal effects that we have previously shown to be dependent on cytochrome P-450 (CYP450) metabolites of aracidonic acid (24) This study evaluated the role of these metabolites in the effects produced by ET-1 on renal blood flow (RBF), cortical blood flow (CBF), medullary blood flow (MBF), and mean arterial blood pressure (MBP). ET-1 (20–200 pmol/kg) increased MBP, renal vascular resistance (RVR), and MBF but reduced CBF and RBF in a dose-dependent manner. The decreases in CBF and RBF, and increases in MBP and RVR were blunted by BMS-182874, an ET a receptor antagonist or BQ-788, an ET b receptor antagonist. Similarly, indomethacin, an inhibitor of cyclooxygenase activity, or 12,12-dibromododecenoic acid (DBDD), a CYP450-dependent inhibitor of production of 20-hydroxyeicosatetraenoic acid (20-HETE), blunted these effects. ET-3 elicited dose-related reduction in CBF and increase in MBF. Indomethacin accentuated the reduction in CBF and attenuated the increase in MBF, as did DBDD. ET-1-induced increase in MBF was attenuated by BQ-788, N ω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide (NO) synthesis, indomethacin, or DBDD. DBDD inhibited the hemodynamic effects ofl-NAME. Miconazole, the inhibitor of CYP450-dependent epoxygenase activity, was without effect. These results indicate that hemodynamic changes produced by ET-1 are mediated by vasoconstrictor prostanoids and/or prostanoid-like substances, possibly, 20-HETE via activation of ET a and ET b receptors. However, the increase in MBF is mediated by vasodilator prostanoids or by NO via ET b receptor activation.

We have previously shown (Ref. 2 ) that endothelium-derived hyperpolarizing factor (EDHF) becomes functional in the fetal ductus arteriosus on removal of nitric oxide and carbon monoxide. From this, it was proposed that EDHF originates from a cytochrome P-450 (CYP450)-catalyzed reaction being inhibited by the two agents. Here, we have examined in the mouse ductus whether EDHF can be identified as an arachidonic acid product of a CYP450 epoxygenase and allied pathways. We did not detect transcripts of the mouse CYP2C subfamily in vessel, while CYP2J subfamily transcripts were expressed with CYP2J6 and CYP2J9. These CYP2J hemoproteins were also detected in the ductus by immunofluorescence microscopy, being colocalized with the endoplasmic reticulum in both endothelial and muscle cells. Distinct CYP450 transcripts were also detected and were responsible for ω-hydroxylation (CYP4A31) and 12R-hydroxylation (CYP4B1). Mass spectrometric analysis showed formation of epoxyeicosatrienoic acids (EETs) in the intact ductus, with 11,12- and 14,15-EETs being more prominent than 5,6- and 8,9-EETs. However, their yield did not increase with nitric oxide/carbon monoxide suppression, nor did it abate with endothelium removal. No evidence was obtained for formation of 12R-hydroxyeicosatrienoic acid and ω-hydroxylation products. 2S-hydroxyeicosatetraenoic acid was instead detected, and, contrary to data implicating this compound as an alternative EDHF, its suppression with baicalein did not modify the EDHF-mediated relaxation to bradykinin. We conclude that none of the more common CYP450-linked arachidonic acid metabolites appears to qualify as EDHF in mouse ductus. We speculate that some novel eicosanoid or a totally unrelated compound requiring CYP450 for its synthesis accounts for EDHF in this vessel.

Abstract Purpose Fatty acid desaturase (FADS) variants associate with fatty acid (FA) and adipose tissue (AT) metabolism and inflammation. Thus, the role of FADS1 variants in the regulation of dietary linoleic acid (LA)-induced effects on AT inflammation was investigated. Methods Subjects homozygotes for the TT and CC genotypes of the FADS1-rs174550 (TT, n = 25 and CC, n = 28) or -rs174547 (TT, n = 42 and CC, n = 28), were either recruited from the METabolic Syndrome In Men cohort to participate in an intervention with LA-enriched diet (FADSDIET) or from the Kuopio Obesity Surgery (KOBS) study. GC and LC–MS for plasma FA proportions and eicosanoid concentrations and AT gene expression for AT inflammatory score (AT-InSc) was determined. Results We observed a diet-genotype interaction between LA-enriched diet and AT-InSc in the FADSDIET. In the KOBS study, interleukin (IL)1 beta mRNA expression in AT was increased in subjects with the TT genotype and highest LA proportion. In the FADSDIET, n-6/LA proportions correlated positively with AT-InSc in those with the TT genotype but not with the CC genotype after LA-enriched diet. Specifically, LA- and AA-derived pro-inflammatory eicosanoids related to CYP450/sEH-pathways correlated positively with AT-InSc in those with the TT genotype, whereas in those with the CC genotype, the negative correlations between pro-inflammatory eicosanoids and AT-InSc related to COX/LOX-pathways. Conclusions LA-enriched diet increases inflammatory AT gene expression in subjects with the TT genotype, while CC genotype could play a protective role against LA-induced AT inflammation. Overall, the FADS1 variant could modify the dietary LA-induced effects on AT inflammation through the differential biosynthesis of AA-derived eicosanoids.