Epoxide Metabolites Research Articles

Identification of structural properties influencing the metabolism of polycyclic aromatic hydrocarbons by cytochrome P450 1A1

Cytochrome P450 1A1 (CYP1A1) has served as a known metabolic enzyme that mediates the carcinogenesis of polycyclic aromatic hydrocarbons (PAHs). However, the structural mechanism involved in the metabolic capacity remains unclear. In this study, thirty-three calculated properties representing the physicochemical and electronic properties of PAH and PAH-CYP1A1 interactions were utilized to identify the key structural properties that affect metabolic processes, including binding ability, metabolic clearance, and mutagenicity, using a quantitative structure-activity relationship (QSAR) strategy combined with docking methods, QM/MM calculations and ab initio calculations. van der Waals interactions (glide vdw) appeared to be important for PAH binding to CYP1A1 and were mainly affected by the molecular weight and hydrophobic structures of PAHs. Interaction features between PAHs and heme, including the distance between iron and carbons of PAHs (Fe_Cmin) and heme vdw, coordinately influence the metabolic clearance of PAHs. Furthermore, the electronic properties (ESP neg variance) appeared to be critical for the mutagenicity of PAHs by CYP1A1 through influencing epoxide metabolite formation. The QSAR models with these key properties provide a new perspective on the structural mechanism of PAH metabolism and provide a useful in silico tool for screening, classifying and predicting PAHs for their metabolism-related toxicities and risk assessment in the environment.

Exploration of binding interaction of aflatoxin metabolite with p53 gene protein, enzymes CYP3A4 in carcinogenesis and its prevention: An in silico study

The foodborne pathogens and their toxic metabolites contaminants are global threat to the human and animal health. Aflatoxin, is one of the fungal carcinogen produced by foodborne pathogen Aspergillus. It metabolizes by cytochrome enzymes and interacts with DNA and p53 gene protein causes cancers in human and animals. In this study, metabolic carcinogenic interactions of aflatoxin, its epoxy metabolite and prevention have explored. This study is stated that epoxide metabolite of aflatoxin more significantly interacts with cytochrome enzyme CYP3A4, human serum protein, p53 gene protein and B-DNA. The observed interacting binding energy of CYP3A4 enzyme was −7.80 kcal/mol and it was bonded with hydrogen bond.Human serum albumin is also observed to be interacted significantly with the epoxide metabolite of aflatoxin through A:HIS146:CA – :UNK1:O22, bond length 2.8958, the binding energy −7.01 kcal/mol and inhibition constant 7.33 uM. The binding interaction of metabolite with B-DNA were A:DA5:H7 – :UNK1:O8, 1.94187, −5.23 kcal/mol and 145.62 µM. The metabolizing enzyme CYP3A4 is inhibited by ketoconazole with less efficiently than betulinic acid and followed by Urolithin b and thymoquinone. Urolithin b a constituent of pomegranate and thymoquinone found in black seed could be a preventive step for the management of aflatoxin mediated toxicity and carcinogenesis. Therefore, in vivo and in vitro laboratory experimentation on inhibition of CYP3A4 enzymes involved in metabolism and epoxidation of toxin by betulinic acid will be further helpful for the aflatoxins detoxification and cancer prevention.

DNA as an in vitro trapping agent for detection of bulky genotoxic metabolites

The instability of electrophilic reactive metabolites in in vitro metabolism studies makes their accurate analysis challenging. To stabilise the reactive compounds prior to their analysis, different trapping agents, such as thiols, amines and cob(I)alamin, have earlier been tested depending on the metabolites to be analysed and the type of study. In the present work, DNA is introduced as a trapping agent for measuring the formation of bulky electrophilic metabolites. Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), was used as a model compound in a rat liver S9 metabolic system. Under physiological incubation conditions, B[a]P metabolises to diol epoxide (BPDE) metabolites which were trapped by DNA resulting in the formation of covalently bound DNA adducts. The methodology for analysis of these adducts included extraction of the DNA from the metabolic system, digestion of the DNA to yield nucleosides and analysis of the BPDE-adduct to deoxyguanosine (BPDE-dG) by liquid chromatography coupled to high resolution mass spectrometry (HRMS). The chromatographic conditions in combination with the high mass accuracy data (±3 ppm) was useful in resolving BPDE-dG in its protonated form from the complex set of ions present in the metabolic matrix. The method was validated in terms of sensitivity, specificity, accuracy, precision and recovery, and applied to provide a preliminary estimate of BPDE-dG levels from the metabolism of B[a]P in rat S9. The use of DNA as a trapping agent for in vitro metabolites has a potential to aid in cancer risk assessment procedure of PAHs, for instance, in inter-species comparison of metabolism to reactive metabolites and can be adapted for screening of genotoxic metabolites, e.g., from emerging environmental contaminants.

Vitamin C inhibits glycidamide-induced genotoxicity and apoptosis in Sertoli cells.

Exposure to the food contaminant acrylamide and its reactive epoxide metabolite glycidamide (GA) induces reactive oxygen species (ROS)-mediated oxidative stress and subsequent cellular death. Recent studies have revealed that the toxic effects of acrylamide may be due to GA, especially on male reproductive system cells. In this regard, it is important to determine the effects of GA on Sertoli cells, which are essential cells for the male reproductive system. Antioxidants should be consumed in sufficient quantities to minimise the effects of environmental pollutants. This study aimed to determine the direct toxic effects of GA and protective effects of vitamin C (VitC) against GA-induced damage in Sertoli cells by measuring cell viability, cytotoxicity, lipid peroxidation, ROS, antioxidant enzyme levels, apoptosis and DNA damage. Sertoli cells were exposed to GA for 24 hours at four different concentrations (ranging between 1 and 1000 μM) and in addition to these GA concentrations to VitC (50 μM). The results of cytotoxicity markers, such as cell viability and lactate dehydrogenase (LDH) showed that GA significantly reduced cell viability and increased LDH levels. We also found that GA induced overproduction of intracellular ROS, increased lipid peroxidation in cellular membrane and triggered cell apoptosis and genotoxicity. In addition, VitC supplementation ameliorated the adverse effects of GA on Sertoli cells. Consequently, these findings suggest that GA may damage the cell function in Sertoli cells, depending on the concentration. Additionally, it was evidenced that VitC has an ameliorative effect on toxicity caused by GA.

Soluble Epoxide Hydrolase Regulation of Lipid Mediators Limits Pain.

The role of lipids in pain signaling is well established and built on decades of knowledge about the pain and inflammation produced by prostaglandin and leukotriene metabolites of cyclooxygenase and lipoxygenase metabolism, respectively. The analgesic properties of other lipid metabolites are more recently coming to light. Lipid metabolites have been observed to act directly at ion channels and G protein-coupled receptors on nociceptive neurons as well as act indirectly at cellular membranes. Cytochrome P450 metabolism of specifically long-chain fatty acids forms epoxide metabolites, the epoxy-fatty acids (EpFA). The biological role of these metabolites has been found to mediate analgesia in several types of pain pathology. EpFA act through a variety of direct and indirect mechanisms to limit pain and inflammation including nuclear receptor agonism, limiting endoplasmic reticulum stress and blocking mitochondrial dysfunction. Small molecule inhibitors of the soluble epoxide hydrolase can stabilize the EpFA in vivo, and this approach has demonstrated relief in preclinical modeled pain pathology. Moreover, the ability to block neuroinflammation extends the potential benefit of targeting soluble epoxide hydrolase to maintain EpFA for neuroprotection in neurodegenerative disease.

Cortex dictamni-induced liver injury in mice: The role of P450-mediated metabolic activation of furanoids

Many furan containing compounds have been reported to be toxic resulted from the metabolic activation of the furan ring to reactive metabolite (RM). Cortex Dictamni (CD), a widely used herbal medicine, has been reported to cause severe even fatal hepatotoxicity. The injurious components and mechanism of CD-induced liver injury remain unclear. Our preliminary study showed that dictamnine, one major furanoid in CD, caused mouse liver injury via its reactive epoxide metabolite. Besides dictamnine, the major components of CD are series of bioactivation-alerting furanoids. Thus, we hypothesize that series of furanoids in CD may undergo metabolic activation and play a key role in CD-induced liver injury. Here, a single oral dose of 60 g/kg ethanol extract of CD (ECD) caused severe hepatocellular necrosis in mice at 24 h post-dose. ECD-induced liver injury showed a dose- and time-dependent manner. The hepatotoxic effects could be completely abolished by P450 nonselective inhibitor 1-aminobenzotriazole (ABT) and strongly modulated by other P450 modulators. The furanoids-concentrated fraction of ECD was responsible for the hepatotoxicity. At least ten furanoids with high abundance in ECD, such as obakunone, dictamnine, fraxinellone, limonin, were found to be metabolized to reactive epoxide or cis-enedione. The RM levels were consistent with the liver injury degree. Multiple furanoids, rather than single one, cooperatively contributed to the hepatotoxicity. ECD-induced liver injury could be reproduced by a mixture of pure furanoids. In summary, this study provides toxic component profiles of CD and demonstrates that P450-mediated bioactivation of multiple furanoids is responsible for CD-induced liver injury.

Mechanism of Chronic Kidney Disease Progression and Novel Biomarkers: A Metabolomic Analysis of Experimental Glomerulonephritis.

While a complex network of cellular and molecular events is known to be involved in the pathophysiological mechanism of chronic kidney disease (CKD), the divergence point between reversal and progression and the event that triggers CKD progression are still unknown. To understand the different mechanisms between reversible and irreversible kidney disease and to search for urinary biomarkers that can predict prognosis, a metabolomic analysis was applied to compare acute and chronic experimental glomerulonephritis (GN) models. Four metabolites, namely, epoxyoctadecenoic acid (EpOME), epoxyeicosatetraenoic acid (EpETE), α-linolenic acid (ALA), and hydroxyretinoic acid, were identified as predictive markers after comparing the chronic nephritis model with acute nephritis and control groups (false discovery rate adjusted p-value (q-value) < 0.05). Renal mRNA expression of cytochrome P450 and epoxide hydrolase was also identified as being involved in the production of epoxide metabolites from these polyunsaturated fatty acids (p < 0.05). These results suggested that the progression of chronic kidney disease is associated with abnormally activated epoxide hydrolase, leading to an increase in EpOME and EpETE as pro-inflammatory eicosanoids.

In Silico Prediction of Metabolic Epoxidation for Drug-like Molecules via Machine Learning Methods.

Epoxidation is one of the reactions in drug metabolism. Since epoxide metabolites would bind with proteins or DNA covalently, drugs should avoid epoxidation metabolism in the body. Due to the instability of epoxide, it is difficult to determine epoxidation experimentally. In silico models based on big data and machine learning methods are hence valuable approaches to predict whether a compound would undergo epoxidation. In this study, we collected 884 epoxidation data manually from various sources, and finally got 829 unique sites of epoxidation. Three types of molecular fingerprints with different lengths (1024, 2048 or 4096 bits) were used to describe the reaction sites. Six machine learning methods were used to build the classification models. The training set and test set were randomly divided into 8 : 2, and 54 models were constructed and evaluated. Four best models were selected for feature selection. The features were then chosen and verified by external validation set. The resulted optimal model had the accuracy and AUC (area under the curve) values at 0.873 and 0.944 for the test set, 0.838 and 0.987 for the external validation set, respectively. The models built in this study could accurately predict whether a compound will undergo epoxidation and which part is most susceptible to epoxidation, which is of great significance for drug design.

Epoxy Fatty Acids Are Promising Targets for Treatment of Pain, Cardiovascular Disease and Other Indications Characterized by Mitochondrial Dysfunction, Endoplasmic Stress and Inflammation.

Bioactive lipid mediators resulting from the metabolism of polyunsaturated fatty acids (PUFA) are controlled by many pathways that regulate the levels of these mediators and maintain homeostasis to prevent disease. PUFA metabolism is driven primarily through three pathways. Two pathways, the cyclooxygenase (COX) and lipoxygenase (LO) enzymatic pathways, form metabolites that are mostly inflammatory, while the third route of metabolism results from the oxidation by the cytochrome P450 enzymes to form hydroxylated PUFA and epoxide metabolites. These epoxygenated fatty acids (EpFA) demonstrate largely anti-inflammatory and beneficial properties, in contrast to the other metabolites formed from the degradation of PUFA. Dysregulation of these systems often leads to chronic disease. Pharmaceutical targets of disease focus on preventing the formation of inflammatory metabolites from the COX and LO pathways, while maintaining the EpFA and increasing their concentration in the body is seen as beneficial to treating and preventing disease. The soluble epoxide hydrolase (sEH) is the major route of metabolism of EpFA. Inhibiting its activity increases concentrations of beneficial EpFA, and often disease states correlate to mutations in the sEH enzyme that increase its activity and decrease the concentrations of EpFA in the body. Recent approaches to increasing EpFA include synthetic mimics that replicate biological activity of EpFA while preventing their metabolism, while other approaches focus on developing small molecule inhibitors to the sEH. Increasing EpFA concentrations in the body has demonstrated multiple beneficial effects in treating many diseases, including inflammatory and painful conditions, cardiovascular disease, neurological and disease of the central nervous system. Demonstration of efficacy in so many disease states can be explained by the fundamental mechanism that EpFA have of maintaining healthy microvasculature and preventing mitochondrial and endoplasmic reticulum stress. While there are no FDA approved methods that target the sEH or other enzymes responsible for metabolizing EpFA, current clinical efforts to test for efficacy by increasing EpFA that include inhibiting the sEH or administration of EpFA mimics that block metabolism are in progress.

Copper (I)-Nicotinate Promotes Gene Expression of CYPs that Produce M1 and Q1 in Aflatoxicosed Rats

Aflatoxin-contaminated food poses a serious risk to both human and animal health. Copper (1)-nicotinate (Cu+-nicotinate) complex potentiated the prophylactic effect against chronic aflatoxicosis in the experimental animals through the synthesis of less toxic metabolites M1 and Q1 which are easily excreted in urine. To investigate the action of the safety Cu+-nicotinate complex on gene expression of Cytochrome 450 (CYP450) system, the liver tissue samples of orally administered rats for 3 weeks with aflatoxin B1 (AFB1; 30 μg/kg body weight), with and without association of the copper complex (400 μg/kg body weight) were assayed for their gene expression of CYP450 families including 1A2, 3A2 and 2C11 as well as histopathological examination of the hepatic tissue samples was performed. The obtained data denoted that the Cu+-nicotinate complex significantly reduced gene expression of CYP2C11 and CYP3A2 that enhancing the most toxic epoxide metabolite. On the contrary, this complex enhanced the expression of CYP1A2 that synthesize the less toxic metabolite M1 and Q1. The histopathological examination mostly confirmed such observation as the signs of aflatoxicosis were absent in Cu+-nicotinate-treated group. Consequently, it could be predicted that the Cu+-nicotinate complex may be medically used as an inhibitory food additive agent against exposure of aflatoxicosis in the intact animals since the complex contains the copper and nicotinic acid, the two daily required biochemical elements for sustaining live in healthy conditions.

In Vitro Biotransformation of the Nrf2 Activator Bardoxolone: Formation of an Epoxide Metabolite That Undergoes Two Novel Glutathione-Mediated Metabolic Pathways: Epoxide Reduction and Oxidative Elimination of Nitrile Moiety

Metabolism of bardoxolone methyl (BARD-Me), an oleanolic acid derivative, and its epoxide metabolite was studied in different in vitro systems. BARD-Me also undergoes glutathione (GSH)-adduct formation via direct nucleophilic attack at the β-carbon of the α,β-unsaturated ketone substituent on the A-ring. The presence of an electron-withdrawing nitrile residue on the α-carbon increases the α,β-unsaturated ketone's susceptibility to nucleophilic attack by thiols. This allows BARD-Me to generate reversible adducts with the thiol groups of cysteine residues in target proteins without the potential toxic liabilities of irreversible covalent adduct formation. However, BARD-Me epoxide can also react with thiols irreversibly. Therefore, the epoxide was synthesized and its metabolic fate studied in vitro. BARD-Me epoxide was found to undergo two novel metabolic biotransformations: epoxide reduction and oxidative elimination of nitrile moiety. Both metabolic pathways proceed via nucleophilic attack of the thiol group of GSH at each of the two carbon atoms of the epoxide as evidenced by the formation of two β-hydroxy sulfide regioisomers. Oxidative elimination of nitrile moiety proceeds via nucleophilic attack of the thiol group of GSH at the epoxide carbon atom that is β to the cyano group to give a cyanohydrin metabolite, which spontaneously decomposes to release cyanide and the corresponding ketone. Nucleophilic attack of the thiol group of GSH at the epoxide carbon atom that is α to the cyano group results in the formation of the GSH monoadduct that undergoes intermolecular reduction with another GSH molecule, followed by elimination of oxidized GSH (GS-SG) and the formation of an enolate intermediate. Upon protonation, the enolate intermediate gives rise to hydroxylated BARD-Me, which is readily converted back to BARD-Me through the elimination of water. The chemical reactivity of the epoxide metabolite and the liberation of cyanide are of significant toxicological interest for the potential utility of BARD-Me as a therapeutic agent.

Pharmacokinetic interaction of brivaracetam on other antiepileptic drugs in adults with focal seizures: Pooled analysis of data from randomized clinical trials

ObjectiveTo assess the effect of brivaracetam (BRV) on steady-state plasma concentrations of commonly prescribed antiepileptic drugs (AEDs). MethodsData were pooled from five randomized, double-blind, placebo-controlled efficacy studies (NCT00175929, NCT00175825, NCT00490035, NCT00464269, and NCT01261325) in which adults with refractory epilepsy, and receiving stable doses of 1–2 AEDs, initiated adjunctive treatment with BRV (or placebo) for up to 12 weeks, following a 4–8 week baseline period. Concentrations of carbamazepine, carbamazepine epoxide, clobazam, clonazepam, lacosamide, lamotrigine, levetiracetam, oxcarbazepine (MHD), phenobarbital, phenytoin, pregabalin, topiramate, valproic acid and zonisamide, were measured during baseline and during BRV or placebo evaluation periods. Log-transformed data for patients receiving BRV dosages of 50–200 mg/day (or placebo) were evaluated using repeated measures analysis of covariance. Geometric least-squares means ratios of respective AED concentrations (treatment vs baseline) and their 90% confidence intervals (CIs) were calculated. Relevant interaction of BRV on the respective AED was inferred if CIs were entirely outside of 0.80–1.25 limits. ResultsWithin the population for analysis (n = 1402), relevant interaction was observed for carbamazepine epoxide alone which increased up to 2-fold from baseline due to inhibition of epoxide hydrolase by BRV, and the effect size was not influenced by concomitant valproic acid. Relevant interaction was not observed for other AEDs. ConclusionIn adults with focal seizures, adjunctive BRV treatment does not affect plasma concentrations of the evaluated AEDs but increases carbamazepine epoxide metabolite. Carbamazepine dose reduction should be considered if tolerability issues arise.

Pharmaceutical Effects of Inhibiting the Soluble Epoxide Hydrolase in Canine Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease that causes pain and bone deterioration driven by an increase in prostaglandins (PGs) and inflammatory cytokines. Current treatments focus on inhibiting prostaglandin production, a pro-inflammatory lipid metabolite, with NSAID drugs; however, other lipid signaling targets could provide safer and more effective treatment strategies. Epoxides of polyunsaturated fatty acids (PUFAs) are anti-inflammatory lipid mediators that are rapidly metabolized by the soluble epoxide hydrolase (sEH) into corresponding vicinal diols. Interestingly, diol levels are increased in the synovial fluid of humans with OA, warranting further research on the biological role of this lipid pathway in the progression of OA. sEH inhibitors (sEHI) stabilize these biologically active, anti-inflammatory lipid epoxides, resulting in analgesia in both neuropathic, and inflammatory pain conditions. Most experimental studies testing the analgesic effects of sEH inhibitors have used experimental rodent models, which do not completely represent the complex etiology of painful diseases. Here, we tested the efficacy of sEHI in aged dogs with natural arthritis to provide a better representation of the clinical manifestations of pain. Two sEHI were administered orally, once daily for 5 days to dogs with naturally occurring arthritis to assess efficacy and pharmacokinetics. Blinded technicians recorded the behavior of the arthritic dogs based on pre-determined criteria to assess pain and function. After 5 days, EC1728 significantly reduced pain at a dose of 5 mg/kg compared to vehicle controls. Pharmacokinetic evaluation showed concentrations exceeding the enzyme potency in both plasma and synovial fluid. In vitro data showed that epoxyeicosatrienoic acid (EETs), epoxide metabolites of arachidonic acid, decreased inflammatory cytokines, IL-6 and TNF-α, and reduced cytotoxicity in canine chondrocytes challenged with IL1β to simulate an arthritic environment. These results provide the first example of altering lipid epoxides as a therapeutic target for OA potentially acting by protecting chondrocytes from inflammatory induced cytotoxicity. Considering the challenges and high variability of naturally occurring disease in aged dogs, these data provide initial proof of concept justification that inhibiting the sEH is a non-NSAID, non-opioid, disease altering strategy for treating OA, and warrants further investigation.

Novel Anti-inflammatory and Vasodilatory ω-3 Endocannabinoid Epoxide Regioisomers.

Accumulating evidence suggests that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) offer protection against vascular inflammation, neuroinflammation, hypertension, and thrombosis. Recently, biochemical studies have demonstrated that these benefits are partially mediated by their conversion to ω-3 endocannabinoid epoxide metabolites. These lipid metabolites originate from the epoxidation of ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by cytochrome P450 (CYP) epoxygenases to form epoxydocosapentaenoic acid-ethanolamides (EDP-EAs) and epoxyeicosatetraenoic acid-ethanolamides (EEQ-EAs), respectively. The EDP-EAs and EEQ-EAs are endogenously produced in rat brain and peripheral organs. Additionally, EDP-EAs and EEQ-EAs dose-dependently decrease pro-inflammatory IL-6 cytokine and increased anti-inflammatory IL-10 cytokine. Furthermore, the EEQ-EAs and EDP-EAs attenuate angiogenesis and cell migration in cancer cells, induce vasodilation in bovine coronary arteries, and reciprocally regulate platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides represent a new class of dual acting molecules that display unique pharmacological properties.

Dictamnine-induced hepatotoxicity in mice: the role of metabolic activation of furan

Cortex Dictamni is extensively used as an herbal medicine worldwide, but is believed to induce hepatotoxicity and even causes mortality in many Asian and European countries. As the most abundant furoquinoline alkaloid ingredient of Cortex Dictamni, dictamnine (DIC) can be metabolically activated by CYP3A to an epoxide metabolite, which possesses the potential to induce hepatotoxicity by covalent binding with proteins. As yet, the hepatotoxicity of DIC and the role played by metabolic activation remain unknown. Here, we found that DIC caused acute liver injury in a time- and dose-dependent manner in mice. The hepatic and urinary DIC epoxide intermediates were observed in DIC-treated mice. Ketoconazole, a CYP3A inhibitor, significantly reduced the hepatotoxicity of DIC and inhibited the formation of reactive metabolites of DIC. Moreover, treatment with 2,3-dihydro-DIC, a DIC analog synthesized by selective reduction of the furan moiety, produced no hepatotoxicity in mice, and no reactive metabolite was formed, suggesting a structural necessity of furan moiety in DIC hepatotoxicity. A time course of gradual hepatic glutathione consumption was observed in DIC-treated mice, while depletion of hepatic glutathione by L-buthionine-S,R-sulfoximine enhanced the hepatotoxicity of DIC. Collectively, this study demonstrates that DIC induces acute hepatocellular injury in mice, and that metabolic activation of furan plays a crucial role in DIC-induced hepatotoxicity.

Antitumorigenic Properties of Omega-3 Endocannabinoid Epoxides.

Accumulating studies have linked inflammation to tumor progression. Dietary omega-3 fatty acids, such as docosahexaenoic acid (DHA), have been shown to suppress tumor growth through their conversion to epoxide metabolites. Alternatively, DHA is converted enzymatically into docosahexaenoylethanolamide (DHEA), an endocannabinoid with antiproliferative activity. Recently, we reported a novel class of anti-inflammatory DHEA-epoxide derivative called epoxydocospentaenoic-ethanolamide (EDP-EA) that contain both ethanolamide and epoxide moieties. Herein, we study the antitumorigenic properties of EDP-EAs in an osteosarcoma (OS) model. First, we show ∼80% increase in EDP-EAs in metastatic versus normal lungs of mice. We found significant differences in the apoptotic and antimigratory potencies of the different EDP-EA regioisomers, which were partially mediated through cannabinoid receptor 1 (CB1). Next, we synthesized derivatives of the most pro-apoptotic regioisomer. These derivatives had reduced hydrolytic susceptibility to fatty acid amide hydrolase (FAAH) and increased CB1-selective binding. Collectively, we report a novel class of EDP-EAs that exhibit antiangiogenic, antitumorigenic, and antimigratory properties in OS.

Metabolite characterization of ambrisentan, in in vitro and in vivo matrices by UHPLC/QTOF/MS/MS: Detection of glutathione conjugate of epoxide metabolite evidenced by in vitro GSH trapping assay

The focus of the present study is on in vitro and in vivo metabolite identification of ambrisentan (AMBR) a selective endothelin type – A (ETA) receptor antagonist using quadruple time-of-flight mass spectrometry (QTOF/MS). in vitro metabolism study was conducted by incubating AMBR in rat liver microsomes (RLM), rat and human liver S9 fractions. In vivo study was carried out through the collection of urine, faeces and plasma samples at various time points after oral administration of AMBR in suspension form at a dose of 25 mg/kg to six male Sprague – Dawley (SD) rats. The samples were prepared using an optimized sample preparation techniques involving protein precipitation (PP), freeze liquid extraction (FLE) and solid phase extraction (SPE). The extracted samples were further concentrated and analyzed by developing a sensitive and specific liquid chromatography-mass spectrometry (LC–MS) method. A total of seventeen metabolites were identified in in vivo samples which includes hydroxyl, demethylated, demethoxylated, hydrolytic, decarboxylated, epoxide and glucuronide metabolites. Most of the metabolites were observed in faeces and urine matrices and few were observed in the plasma matrix. Only ten metabolites were identified in in vitro study which was commonly observed in in vivo study. The detailed structural elucidation of all the metabolites was done using UHPLC/QTOF/MS/MS in combination with accurate mass measurements. The toxicity profile of AMBR and its metabolites were predicted using TOPKAT software. In addition, a mass spectrometric method was developed for the detection and characterization of GSH-trapped reactive epoxide metabolitein human liver S9 fraction supplemented with glutathione (GSH) as trapping agent.

Inhibition of pancreatic carcinoma growth through enhancing epoxide metabolic profile of ω‐3 polyunsaturated fatty acid by inhibiting soluble epoxide hydrolase

Anti‐inflammatory and anti‐carcinogenic effects of ω‐3 polyunsaturated fatty acid (PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), etc., are well known; but the mechanism/s remains unclear. Of the three metabolic pathways {Cyclooxygenase (COX), Lipooxygenase (LOX) and Cyto‐p450 (CYP)}, ω‐3 PUFAs are predominantly metabolized by CYP epoxygenase/s, leading to an accumulation of ω‐3 epoxy fatty acid (ω‐3 epoxides); and ω‐3 PUFAs are poor substrates of COX and LOX. However, under physiologic conditions, these ω‐3 epoxides are quickly inactivated by soluble epoxide hydrolase (sEH) to the diol products, and a sEH inhibitor appears crucial to stabilizing/enhancing these ω‐3 epoxides actions. In this study, using a transplantable murine PK03 pancreatic carcinoma model which carried mutant Kras gene, we have performed three animal experiments using Fat‐1 transgenic, sEH (−/−), and C57BL/6J mice subcutaneously implanted with a murine PK03 pancreatic carcinoma cells treated with or without sEH inhibitor and fed with either AIN‐76A or AIN‐76A diet supplemented with ω‐3 PUFAs. Aims of this study are to determine 1) if sEH gene knockout or inhibition stabilize the epoxide metabolites of ω‐3/ω‐6 PUFAs, 2) if sEH gene knockout or inhibition has more potent effects on inhibiting pancreatic cancer growth via enhancing the ω‐3 epoxide metabolites, and 3) potential molecular targets on mutant Kras mediated signaling, angiogenesis and inflammation. The significant findings were summarized as follows: in Fat‐1 mice treated with sEH inhibitor t‐AUCB or sEH (−/−) mice fed with AIN‐76A diet supplemented with ω‐3 PUFAs or C57BL/6J mice treated with sEH inhibitor and AIN‐76A diet supplemented with ω‐3 PUFAs, compared to fat‐1 mice without t‐AUCB treatment or sEH(−/−) mice fed with AIN‐76A diet, or C57BL/6J mice fed AIN‐76A diet, i) the size and tumor volume of xenograft PK03 pancreatic carcinoma was significantly inhibited ii) more glandular formation/well differentiation in the treated PK03 pancreatic carcinoma was observed by histopathologic analysis, iii) significant less inflammatory cell infiltrates and cell proliferation labeled immunohistochemically by anti‐myeloperoxidase and ki‐67 antibodies, as well as significant increased caspase‐3 labeled apoptosis and significant reduced intratumoral angiogenesis labeled by CD34 antibody were observed in the treated PK03 pancreatic carcinoma; iv) Western blot analysis showed a significant inhibition of mutant Kras‐activated signals including phosphorylated cRaf, MEK and ERK. Eicosanoic acid metabolic profiling analyzed by LC/MSMS method revealed a significant increase of the ratios of Epoxy to DiHDPE for DHA and EPA, and Epoxy/DiHOME for ARA and LA, as well as a significant increase of epoxy metabolites of DHA, EPA, ARA and LA. These results indicate that ω‐3 PUFAs supplementation together with sEH inhibition plays an important role in inhibiting pancreatic carcinogenesis mainly via enhancing the epoxy metabolites, and are highly potential approach for preventing pancreatic cancer.Support or Funding InformationSupported by NIH R01 DK107767 to Dr. Guang‐Yu Yang.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Structural‐Activity Features of Long Chain Polyunsaturated Fatty Acid Epoxides in Angiogenesis

The cytochrome P450‐derived fatty acid epoxides are bioactive lipids with regulatory roles in a wide range of homeostatic and pathophysiological events. These lipid mediators are metabolized efficiently by the mammalian soluble epoxide hydrolase enzyme (sEH) to corresponding 1,2‐diols. Inhibition of sEH has been shown to be beneficial to human health via increasing concentrations of a variety of short‐lived epoxylipids in vivo (2). Identifying the most potent polyunsaturated fatty acid (PUFA) epoxide metabolites will provide insight on the effects of sEH inhibition. Recent studies have shown that the omega‐6 epoxyeicosatrienoic acids (EETs) from arachidonic acid are pro‐angiogenic, while the omega‐3, docosahexaenoic acid (DHA)‐derived epoxydocosapentaenoic acids (EDPs) inhibit angiogenesis, tumor growth, and metastasis (1). Given these findings, we are interested in exploring the roles of various long chain PUFA epoxides in angiogenesis. We tested a library of omega‐3 and omega‐6 epoxides in a newly developed high‐throughput tube formation assay and found their effects are regiospecific and stereospecific; Of note, the 19(S),20(R)‐EDP stereoisomer was a less effective inhibitor of endothelial cell tube formation than its respective racemic mixture. Here, we report the angiogenic activities of diverse omega‐3 and omega‐6 fatty acid epoxides that could be present in mammals from the diet, including the less explored epoxides from both omega‐3 and omega‐6 docosapentaenoic acid (DPA). We will discuss the structure‐activity features of the most potent epoxide species and also present methodology to synthesize and purify the regioisomers and stereoisomers of each series of epoxides. Ultimately, understanding activities of sEH substrates in specific regulatory environments will guide development of targeted sEH modulators that promote the beneficial effects of PUFA metabolites in human health and disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of an Epoxide Metabolite of Lycopene in Human Plasma Using 13C-Labeling and QTOF-MS.

The carotenoid lycopene is a bioactive component of tomatoes and is hypothesized to reduce risk of several chronic diseases, such as prostate cancer. The metabolism of lycopene is only beginning to be understood and some studies suggest that metabolites of lycopene may be partially responsible for bioactivity associated with the parent compound. The detection and characterization of these compounds in vivo is an important step in understanding lycopene bioactivity. The metabolism of lycopene likely involves both chemical and enzymatic oxidation. While numerous lycopene metabolites have been proposed, few have actually been identified in vivo following lycopene intake. Here, LC-QTOF-MS was used along with 13C-labeling to investigate the post-prandial oxidative metabolism of lycopene in human plasma. Previously reported aldehyde cleavage products were not detected, but a lycopene 1,2-epoxide was identified as a new candidate oxidative metabolite.