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Obesity induces chronic inflammation resulting in insulin resistance and metabolic disorders. Cold exposure can improve insulin sensitivity in humans and rodents, but the mechanisms have not been fully elucidated. Here, we find that cold resolves obesity-induced inflammation and insulin resistance and improves glucose tolerance in diet-induced obese mice. The beneficial effects of cold exposure on improving obesity-induced inflammation and insulin resistance depend on brown adipose tissue (BAT) and liver. Using targeted liquid chromatography with tandem mass spectrometry, we discovered that cold and β3-adrenergic stimulation promote BAT to produce maresin 2 (MaR2), a member of the specialized pro-resolving mediators of bioactive lipids that play a role in the resolution of inflammation. Notably, MaR2 reduces inflammation in obesity in part by targeting macrophages in the liver. Thus, BAT-derived MaR2 could contribute to the beneficial effects of BAT activation in resolving obesity-induced inflammation and may inform therapeutic approaches to combat obesity and its complications.

Brown adipose tissue is a thermogenic organ that possesses anti-diabetic and anti-obesogenic potential. There has recently been growing interest on the secretory role of brown adipose tissue in regulating whole-body metabolism. Several signaling lipids, including 12-HEPE and 12,13-diHOME, have been shown to be secreted by brown adipose tissue and have demonstrated roles in regulating whole-body energy metabolism. Lipidomics platforms that broadly characterize the signaling lipidome can deconvolute the underlying biology of the lipid metabolites having a broad systemic impact on physiology. Herein, we describe how to perform and analyze LC-MS/MS signaling lipidomics on mature brown adipocytes.

Epoxyeicosatrienoic acids (EETs) are formed from the metabolism of arachidonic acid by cytochrome P450s. EETs promote angiogenesis linked to tumor growth in various cancer models that is attenuated in vivo by cyclooxygenase 2 (COX-2) inhibitors. This study further defines a role for COX-2 in mediating endothelial EET metabolism promoting angiogenesis. Using human aortic endothelial cells (HAECs), we quantified 8,9-EET-induced tube formation and cell migration as indicators of angiogenic potential in the presence and absence of a COX-2 inducer [phorbol 12,13-dibutyrate (PDBu)]. The angiogenic response to 8,9-EET in the presence of PDBu was 3-fold that elicited by 8,9-EET stabilized with a soluble epoxide hydrolase inhibitor (t-TUCB). Contributing to this response was the COX-2 metabolite of 8,9-EET, the 11-hydroxy-8,9-EET (8,9,11-EHET), which exogenously enhanced angiogenic responses in HAECs at levels comparable to those elicited by vascular endothelial growth factor (VEGF). In contrast, the 15-hydroxy-8,9-EET isomer was also formed but inactive. The 8,9,11-EHET also promoted expression of the VEGF family of tyrosine kinase receptors. These results indicate that 8,9-EET-stimulated angiogenesis is enhanced by COX-2 metabolism in the endothelium through the formation of 8,9,11-EHET. This alternative pathway for the metabolism of 8,9-EET may be particularly important in regulating angiogenesis under circumstances in which COX-2 is induced, such as in cancer tumor growth and inflammation.

Fatty acid amide hydrolase (FAAH) is a membrane protein that hydrolyzes endocannabinoids, and its inhibition produces analgesic and anti-inflammatory effects. The soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids. EETs have anti-inflammatory and inflammation resolving properties, thus inhibition of sEH consequently reduces inflammation. Concurrent inhibition of both enzymes may represent a novel approach in the treatment of chronic pain. Drugs with multiple targets can provide a superior therapeutic effect and a decrease in side effects compared to ligands with single targets. Previously, microwave-assisted methodologies were employed to synthesize libraries of benzothiazole analogs from which high affinity dual inhibitors (e.g. 3, sEH IC50=9.6nM; FAAH IC50=7nM) were identified. Here, our structure-activity relationship studies revealed that the 4-phenylthiazole moiety is well tolerated by both enzymes, producing excellent inhibition potencies in the low nanomolar range (e.g. 6o, sEH IC50=2.5nM; FAAH IC50=9.8nM). Docking experiments show that the new class of dual inhibitors bind within the catalytic sites of both enzymes. Prediction of several pharmacokinetic/pharmacodynamic properties suggest that these new dual inhibitors are good candidates for further in vivo evaluation. Finally, dual inhibitor 3 was tested in the Formalin Test, a rat model of acute inflammatory pain. The data indicate that 3 produces antinociception against the inflammatory phase of the Formalin Test in vivo and is metabolically stable following intraperitoneal administration in male rats. Further, antinociception produced by 3 is comparable to that of ketoprofen, a traditional nonsteroidal anti-inflammatory drug. The results presented here will help toward the long-term goal of developing novel non-opioid therapeutics for pain management.

Epoxy-fatty acids (EpFAs) are endogenous lipid mediators that have a large breadth of biological activities, including the regulation of blood pressure, inflammation, angiogenesis, and pain perception. For the past 20 years, soluble epoxide hydrolase (sEH) has been recognized as the primary enzyme for degrading EpFAs in vivo. The sEH converts EpFAs to the generally less biologically active 1,2-diols, which are quickly eliminated from the body. Thus, inhibitors of sEH are being developed as potential drug therapeutics for various diseases including neuropathic pain. Recent findings suggest that other epoxide hydrolases (EHs) such as microsomal epoxide hydrolase (mEH) and epoxide hydrolase-3 (EH3) can contribute significantly to the in vivo metabolism of EpFAs. In this study, we used two complementary approaches to probe the relative importance of sEH, mEH, and EH3 in 15 human tissue extracts: hydrolysis of 14,15-EET and 13,14-EDP using selective inhibitors and protein quantification. The sEH hydrolyzed the majority of EpFAs in all of the tissues investigated, mEH hydrolyzed a significant portion of EpFAs in several tissues, whereas no significant role in EpFAs metabolism was observed for EH3. Our findings indicate that residual mEH activity could limit the therapeutic efficacy of sEH inhibition in certain organs.

Linoleic acid (LA) is the most abundant polyunsaturated fatty acid found in the western diet. Cytochrome P450-derived LA metabolites 9,10-epoxyoctadecenoic acid (9,10-EpOME), 12,13-epoxyoctadecenoic acid (12,13-EpOME), 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) and 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME) have been studied for their association with various disease states and biological functions. Previous studies of the EpOMEs and DiHOMEs have focused on their roles in cytotoxic processes, primarily in the inhibition of the neutrophil respiratory burst. More recent research has suggested the DiHOMEs may be important lipid mediators in pain perception, altered immune response, and brown adipose tissue (BAT) activation by cold and exercise. The purpose of this review is to summarize the current understanding of the physiological and pathophysiological roles and modes of action of the EpOMEs and DiHOMEs in health and disease.

Multitarget-directed ligands are a promising class of drugs for discovering innovative new therapies for difficult to treat diseases. In this study, we designed dual inhibitors targeting the human fatty acid amide hydrolase (FAAH) enzyme and human soluble epoxide hydrolase (sEH) enzyme. Targeting both of these enzymes concurrently with single target inhibitors synergistically reduces inflammatory and neuropathic pain; thus, dual FAAH/sEH inhibitors are likely to be powerful analgesics. Here, we identified the piperidinyl-sulfonamide moiety as a common pharmacophore and optimized several inhibitors to have excellent inhibition profiles on both targeted enzymes simultaneously. In addition, several inhibitors show good predicted pharmacokinetic properties. These results suggest that this series of inhibitors has the potential to be further developed as new lead candidates and therapeutics in pain management.

Increasing biological thermogenesis (the conversion of chemical energy to heat) has been proposed as an approach towards lowering blood glucose, improving insulin sensitivity and mitigating type 2 diabetes. Non-shivering thermogenesis occurs primarily in adipocytes that express uncoupling protein 1 (UCP1) including brown adipose tissue (BAT) and beige adipose tissue but does not substantially occur in white adipose tissue (WAT). To identify genes that regulate thermogenesis and BAT function, we recently compared genes expressed in paired human patient samples (n = 10) of the deep neck BAT located next to the carotid sheath and superficial subcutaneous WAT in the neck. We found UDP-glucuronide transferase 1A (UGT1A) is co-expressed in individuals with a high amount of the thermogenic protein uncoupling protein 1 (UCP1). This enzyme is known to glucuronidate metabolites, including bilirubin and xenobiotics, which increases their water solubility and excretion. Based on this function, we hypothesize UGT1A regulates BAT not through direct action of the enzyme but through modulating the availability of thermogenic molecules. Here, we characterized expression of different UGT1A isoforms in vivo and in vitro. Cold exposure is an effective method for activating BAT and increasing thermogenesis in mice. Interestingly, expression of multiple UGT1A isoforms, including UGT1A6a and UGT1A6b, are decreased in BAT of mice exposed to a cold environment (5°C) for 7 days. This finding is consistent with data in multiple publicly available datasets which have shown UGT1A6a/6b are decreased in cold exposure and other thermogenic stimuli. In vitro, most UGT1A isoforms were increased during the differentiation of murine brown adipocytes but only certain isoforms were differentially expressed during in vitro differentiation in human brown adipocytes. Based on these data, we propose that UGT1A may have a function related to thermogenesis and understanding its function could lead to novel approaches for activating BAT. Disclosure S. Kodani: None. M. Lynes: None. F. Shamsi: None. C. Wang: None. S. Sugimoto: None. R. Xue: None. T. Huang: None. A.P. White: None. A.M. Cypess: None. Y. Tseng: Consultant; Self; Boehringer Ingelheim International GmbH. Funding National Institutes of Health (R01DK07709, R01DK102898, T32DK007260)

The veterinary pharmacopeia available to treat pain and inflammation is limited in number, target of action and efficacy. Inhibitors of soluble epoxide hydrolase (sEH) are a new class of anti-inflammatory, pro-resolving and analgesic drugs being tested in humans that have demonstrated efficacy in laboratory animals. They block the hydrolysis, and thus, increase endogenous concentrations of analgesic and anti-inflammatory signaling molecules called epoxy-fatty acids. Here, we screened a library of 2,300 inhibitors of the sEH human against partially purified feline, canine and equine hepatic sEH to identify inhibitors that are broadly potent among species. Six very potent sEH inhibitors (IC50 < 1 nM for each enzyme tested) were identified. Their microsomal stability was then measured in hepatic extracts from cat, dog and horse, as well as their solubility in solvents suitable for the formulation of drugs. The trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB, 1,728) appears to be the best compromise between stability and potency across species. Thus, it was selected for further testing in veterinary clinical trials of pain and inflammation in animals.

In this issue of Developmental Cell, Nguyen et al. use scRNA-seq to explore the changing cellular landscape of subcutaneous adipose tissue during aging. In the process, they discover a new population of cells called age-dependent regulatory cells (ARC), which contributes to age-related adipose tissue dysfunction that drives metabolic disease.

Carboxylesterases are well known for their role in the metabolism of xenobiotics. However, recent studies have also implicated carboxylesterases in regulating a number of physiological processes including metabolic homeostasis and macrophage development, underlying the need to quantify them individually. Unfortunately, current methods for selectively measuring the catalytic activity of individual carboxylesterases are not sufficiently sensitive to support many biological studies. In order to develop a more sensitive and selective method to measure the activity of human carboxylesterase 1 (hCE1), we generated and tested novel substrates with a fluorescent aminopyridine leaving group. hCE1 showed at least a 10-fold higher preference for the optimized substrate 4-MOMMP than the 13 other esterases tested. Because of the high stability of 4-MOMMP and its hydrolysis product, this substrate can be used to measure esterase activity over extended incubation periods yielding a low picogram (femtomol) limit of detection. This sensitivity is comparable to current ELISA methods; however, the new assay quantifies only the catalytically active enzyme facilitating direct correlation to biological processes. The method described herein may allow hCE1 activity to be used as a biomarker for predicting drug pharmacokinetics, early detection of hepatocellular carcinoma, and other disease states where the activity of hCE1 is altered.

Pulmonary arterial hypertension is a complication of methamphetamine use (METH-PAH), but the pathogenic mechanisms are unknown. Given that cytochrome P450 2D6 (CYP2D6) and carboxylesterase 1 (CES1) are involved in metabolism of METH and other amphetamine-like compounds, we postulated that loss of function variants could contribute to METH-PAH. Although no difference in CYP2D6 expression was seen by lung immunofluorescence, CES1 expression was significantly reduced in endothelium of METH-PAH microvessels. Mass spectrometry analysis showed that healthy pulmonary microvascular endothelial cells (PMVECs) have the capacity to both internalize and metabolize METH. Furthermore, whole exome sequencing data from 18 METH-PAH patients revealed that 94.4% of METH-PAH patients were heterozygous carriers of a single nucleotide variant (SNV; rs115629050) predicted to reduce CES1 activity. PMVECs transfected with this CES1 variant demonstrated significantly higher rates of METH-induced apoptosis. METH exposure results in increased formation of reactive oxygen species (ROS) and a compensatory autophagy response. Compared with healthy cells, CES1-deficient PMVECs lack a robust autophagy response despite higher ROS, which correlates with increased apoptosis. We propose that reduced CES1 expression/activity could promote development of METH-PAH by increasing PMVEC apoptosis and small vessel loss.

OBJECTIVES: Brown adipose tissue has recently emerged as a novel target for obesity treatment and prevention. In contrast to the lipid storing function of white adipocytes, brown adipocytes are responsible for dissipating energy as heat, a process involving uncoupling protein 1 (UCP1). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme that converts epoxy fatty acids (EpFAs) into less active diols. By stabilizing endogenous EpFAs, potent small molecule sEH inhibitors have been shown to be beneficial for many chronic diseases. Several recent papers have reported that sEH inhibitors are able to reduce diet-induced obesity, possibly by upregulating UCP1 expression. In the current study, we sought to study the mechanisms by which sEH inhibitor acts on brown preadipocytes. METHODS: The effects of a potent sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), on murine brown adipocyte differentiation were evaluated by lipid accumulation and expression of brown adipocyte marker genes. PPAR alpha and PPAR gamma activation by t-AUCB was measured by their respective transactivation assays. The roles of PPARs were further studied by pharmacological antagonism and knockdown experiments by small RNA interference. RESULTS: We report that sEH expression was increased during murine brown adipocyte differentiation. t-AUCB dose-dependently promoted brown adipocyte differentiation. Moreover, we demonstrate that t-AUCB activated PPAR alpha, but not PPAR gamma. t-AUCB-induced upregulation of thermogenic gene Ucp1 and Pgc1 alpha and the general differentiation marker Fabp4 were significantly attenuated by the antagonist of PPAR alpha, GW6471. In contrast, they were only partially attenuated by the antagonist of PPAR gamma, GW9662, and specific knockdown of PPAR gamma. CONCLUSIONS: Our findings suggest that sEH may regulate brown adipogenesis and sEH pharmacological inhibition by t-AUCB promotes brown adipogenesis, possibly through activation of PPAR alpha. FUNDING SOURCES: The work is supported by NIH 1R15DK114790-01A1 (to LZ), R00DK100736 (to AB) and R01ES002710 (to BDH).

Adaptive thermogenesis is a catabolic process that consumes energy-storing molecules and expends that energy as heat in response to environmental changes. This process occurs primarily in brown and beige adipose tissue. Thermogenesis is regulated by many factors, including lipid derived paracrine and endocrine hormones called lipokines. Recently, technologic advances for identifying new lipid biomarkers of thermogenic activity have shed light on a diverse set of lipokines that act through different pathways to regulate energy expenditure. In this review, we highlight a few examples of lipokines that regulate thermogenesis. The biosynthesis, regulation, and effects of the thermogenic lipokines in several families are reviewed, including oloeylethanolamine, endocannabinoids, prostaglandin E2, and 12,13-diHOME. These thermogenic lipokines present potential therapeutic targets to combat states of excess energy storage, such as obesity and related metabolic disorders.

Neuroinflammation is a physiologic response aimed at protecting the central nervous system during injury. However, unresolved and chronic neuroinflammation can lead to long term damage and eventually neurologic disease including Parkinson's disease, Alzheimer's disease and dementia. Recently, enhancing the concentration of epoxyeicosatrienoic acids (EETs) through blocking their hydrolytic degradation by soluble epoxide hydrolase (sEH) has been applied towards reducing the long-term damage associated with central neurologic insults. Evidence suggests this protective effect is mediated, at least in part, through polarization of microglia to an anti-inflammatory phenotype that blocks the inflammatory actions of prostaglandins and promotes wound repair. This mini-review overviews the epidemiologic basis for using sEH inhibition towards neuroinflammatory disease and pharmacologic studies testing sEH inhibition in several neurologic diseases. Additionally, the combination of sEH inhibition with other eicosanoid signaling pathways is considered as an enhanced approach for developing potent neuroprotectants.

Adamantyl-containing 1,3-disubstituted ureas, bisureas, and biscarbamates containing different spacers between the ureylene or the carbamate groups and the adamantyl radical were synthesized. Their inhibitory activity against soluble epoxide hydrolases of mammals and humans (sEH, E.C. 3.3.2.10) was studied. The compounds were found to possess high inhibitory activity on the level of 0.8—2.7 nmol L–1. A relationship between the inhibitor structure and its activity was established.

Indomethacin, a nonsteroidal anti-inflammatory drug, has been shown to induce white adipocyte differentiation; however, its roles in brown adipocyte differentiation and activation in brown adipose tissue (BAT) and obesity are unknown. To address this issue, we treated mouse brown preadipocytes with different doses of indomethacin, and delivered indomethacin to interscapular BAT (iBAT) of obese mice using implanted osmotic pumps. Indomethacin dose dependently increased brown preadipocyte differentiation and upregulated both mRNA and protein expression of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor (PPAR) γ coactivator 1-alpha. The mechanistic study showed that indomethacin significantly activated the reporter driven by the PPAR response element, indicating that indomethacin may work as a PPARγ agonist in this cell line. Consistently, indomethacin significantly decreased iBAT mass and fasting blood glucose levels in high-fat diet–induced obesity (DIO) mice. Histologic analysis showed that brown adipocytes of indomethacin-treated mice contained smaller lipid droplets compared with control mice, suggesting that indomethacin alleviated the whitening of BAT induced by the high-fat diet. Moreover, indomethacin significantly increased UCP1 mRNA expression in iBAT. Taken together, this study indicates that indomethacin can promote mouse brown adipocyte differentiation, and might increase brown fat and glucose oxidation capacity in DIO mice.

Multi-target inhibitors have become increasing popular as a means to leverage the advantages of poly-pharmacology while simplifying drug delivery. Here, we describe dual inhibitors for soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH), two targets known to synergize when treating inflammatory and neuropathic pain. The structure activity relationship (SAR) study described herein initially started with t-TUCB (trans-4-[4-(3-trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid), a potent sEH inhibitor that was previously shown to weakly inhibit FAAH. Inhibitors with a 6-fold increase of FAAH potency while maintaining high sEH potency were developed by optimization. Interestingly, compared to most FAAH inhibitors that inhibit through time-dependent covalent modification, t-TUCB and related compounds appear to inhibit FAAH through a time-independent, competitive mechanism. These inhibitors are selective for FAAH over other serine hydrolases. In addition, FAAH inhibition by t-TUCB appears to be higher in human FAAH over other species; however, the new dual sEH/FAAH inhibitors have improved cross-species potency. These dual inhibitors may be useful for future studies in understanding the therapeutic application of dual sEH/FAAH inhibition.

The cyclooxygenase-2 (COX-2) selective inhibitor celecoxib is widely used in the treatment of pain and inflammation. Celecoxib has been explored as a possible treatment of liver fibrosis with contradictory results, depending on the model. The present study reports the effect of celecoxib in a 5-week carbon tetrachloride (CCl(4))-induced liver fibrosis mouse model. Celecoxib alone and in combination with inhibitors of the enzyme-soluble epoxide hydrolase (sEH), as well as a dual inhibitor that targets both COX-2 and sEH, were administered via osmotic minipump to mice receiving intraperitoneal injections of CCl(4). Collagen deposition was elevated in the mice treated with both celecoxib and CCl(4) compared with the control or CCl(4)-only groups, as assessed by trichrome staining. Histopathology revealed more extensive fibrosis and cell death in the animals treated with both celecoxib and CCl(4) compared with all other experimental groups. Although some markers of fibrosis, such as matrix metalloprotease, were unchanged or lowered in the animals treated with both celecoxib and CCl(4), overall, hepatic fibrosis was more severe in this group. Cotreatment with celecoxib and an inhibitor of sEH or treatment with a dual inhibitor of COX-2 and sEH decreased the elevated levels of fibrotic markers observed in the group that received both celecoxib and CCl(4). Oxylipid analysis revealed that celecoxib reduced the level of prostaglandin E(2) relative to the CCl(4) only group. Overall, celecoxib treatment did not decrease liver fibrosis in CCl(4)-treated mice.

We have previously identified and reported several potent piperidine-derived amide inhibitors of the human soluble epoxide hydrolase (sEH) enzyme. The inhibition of this enzyme leads to elevated levels of epoxyeicosatrienoic acids (EETs), which are known to possess anti-inflammatory, vasodilatory, and anti-fibrotic effects. Herein, we report the synthesis of 9 analogs of the lead sEH inhibitor and the follow-up structure-activity relationship and liver microsome stability studies. Our findings show that isosteric modifications that lead to significant alterations in the steric and electronic properties at a specific position in the molecule can reduce the efficacy by up to 75-fold. On the other hand, substituting hydrogen with deuterium produces a notable increase (∼30%) in the molecules' half-lives in both rat and human microsomes, while maintaining sEH inhibition potency. These data highlight the utility of isosteric replacement for improving bioavailability, and the newly-synthesized inhibitor structures may thus, serve as a starting point for preclinical development. Our docking study reveals that in the catalytic pocket of sEH, these analogs are in proximity of the key amino acids involved in hydrolysis of EETs.

Vehicle exhaust is rich in polycyclic aromatic hydrocarbons (PAH) and can be a dominant contributor to ultrafine urban particulate matter (PM). Exposure to ultrafine PM is correlated with respiratory infections and asthmatic symptoms in young children. The lung undergoes substantial growth, alveolarization, and cellular maturation within the first years of life, which may be impacted by environmental pollutants such as PM. PAHs in PM can serve as ligands for the aryl hydrocarbon receptor (AhR) that induces expression of certain isozymes in the cytochrome P-450 superfamily, such as CYP1A1 and CYP1B1, localized in specific lung cell types. Although AhR activation and induction has been widely studied, its context within PM exposure and impact on the developing lung is poorly understood. In response, we have developed a replicable ultrafine premixed flame particle (PFP) generating system and used in vitro and in vivo models to define PM effects on AhR activation in the developing lung. We exposed 7-day neonatal and adult rats to a single 6-h PFP exposure and determined that PFPs cause significant parenchymal toxicity in neonates. PFPs contain weak AhR agonists that upregulate AhR-xenobiotic response element activity and expression and are capable inducers of CYP1A1 and CYP1B1 expression in both ages with different spatial and temporal patterns. Neonatal CYP1A1 expression was muted and delayed compared with adults, possibly because of differences in the enzyme maturation. We conclude that the inability of neonates to sufficiently adapt in response to PFP exposure may, in part, explain their susceptibility to PFP and urban ultrafine PM.

The plant growth regulator ethephon (2-chloroethylphosphonic acid) inhibits human butyrylcholinesterase (BChE) by making a covalent adduct on the active site serine 198. Our goal was to extend earlier studies on ethephon inhibition. Addition of freshly prepared ethephon to BChE in buffered medium, at pH 7.0 and 22 °C, resulted in no inhibition initially. However, inhibition developed progressively over 60 min of incubation. Preincubation of ethephon in pH 7-9 buffers increased its initial inhibitory potency. These observations indicated that ethephon itself was not the inhibitor. About 3% of the initial ethephon could be trapped as a BChE adduct. Mass spectral analysis of the active site peptide from inhibited BChE showed that the inhibitor added a mass of 108 Da to the active site serine on peptide FGES198AGAAS. This result rules out a previous hypothesis that ethephon adds HPO3 to BChE (added mass of 80 Da). To accommodate these observations, we propose that in aqueous media at neutral to slightly alkaline pH about 3% of the ethephon is converted (t1/2 = 9.9 h at pH 7.0) into a cyclic oxaphosphetane which is the actual BChE inhibitor forming the 2-hydroxyethylphosphonate adduct on BChE at Ser198 while about 97% of the ethephon breaks down to ethylene (t1/2 = 11-48 h at pH 7.0) which is responsible for plant growth regulation.

Parabens are a class of small molecules that are regularly used as preservatives in a variety of personal care products. Several parabens, including butylparaben and benzylparaben, have been found to interfere with endocrine signaling and to stimulate adipocyte differentiation. We hypothesized these biological effects could be due to interference with the endocannabinoid system and identified fatty acid amide hydrolase (FAAH) as the direct molecular target of parabens. FAAH inhibition by parabens yields mixed-type and time-independent kinetics. Additionally, structure activity relationships indicate FAAH inhibition is selective for the paraben class of compounds and the more hydrophobic parabens have higher potency. Parabens enhanced 3T3-L1 adipocyte differentiation in a dose dependent fashion, different from two other FAAH inhibitors URB597 and PF622. Moreover, parabens, URB597 and PF622 all failed to enhance AEA-induced differentiation. Furthermore, rimonabant, a cannabinoid receptor 1 (CB1)-selective antagonist, did not attenuate paraben-induced adipocyte differentiation. Thus, adipogenesis mediated by parabens likely occurs through modulation of endocannabinoids, but cell differentiation is independent of direct activation of CB1 by endocannabinoids.

Exposure to the halogenated hydrocarbon carbon tetrachloride (CCl4) leads to hepatic lipid peroxidation, inflammation, and fibrosis. Dietary supplementation of ω-3 fatty acids has been increasingly advocated as being generally anti-inflammatory, though its effect in models of liver fibrosis is mixed. This raises the question of whether diets high in ω-3 fatty acids will result in a greater sensitivity or resistance to liver fibrosis as a result of environmental toxicants like CCl4. In this study we fed CCl4-treated mice a high ω-3 diet (using a mix of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) ethyl esters). We also co-administered an inhibitor of soluble epoxide hydrolase, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which has been shown to boost anti-inflammatory epoxy fatty acids that are produced from both ω-3 and ω-6 dietary lipids. In this study, we showed that soluble epoxide inhibitors reduced CCl4-induced liver fibrosis. Three major results were obtained. First, the ω-3 enriched diet did not attenuate CCl4-induced liver fibrosis as judged by collagen deposition and collagen mRNA expression. Second, the ω-3 enriched diet raised hepatic tissue levels of several inflammatory lipoxygenase metabolites and prostaglandins, including PGE2. Third, treatment with TPPU in drinking water in conjunction with the ω-3 enriched diet resulted in a reduction in liver fibrosis compared to all other groups. Taken together, these results indicate that dietary ω-3 supplementation alone did not attenuate CCl4-induced liver fibrosis. Additionally, oxylipin signaling molecules may play role in the CCl4-induced liver fibrosis in the high ω-3 diet groups.

Proton pump inhibitors such as omeprazole (OME) reduce the severity of gastrointestinal (GI) ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs) but can also increase the chance of dysbiosis. The aim of this study was to test the hypothesis that preventive use of a soluble epoxide hydrolase inhibitor (sEHI) such as TPPU can decrease NSAID-induced ulcers by increasing anti-inflammatory epoxyeicosatrienoic acids (EETs). Dose- [10, 30, and 100 mg/kg, by mouth (PO)] and time-dependent (6 and 18 hours) ulcerative effects of diclofenac sodium (DCF, an NSAID) were studied in the small intestine of Swiss Webster mice. Dose-dependent effects of TPPU (0.001-0.1 mg/kg per day for 7 days, in drinking water) were evaluated in DCF-induced intestinal toxicity and compared with OME (20 mg/kg, PO). In addition, the effect of treatment was studied on levels of Hb in blood, EETs in plasma, inflammatory markers such as myeloperoxidase (MPO) in intestinal tissue homogenates, and tissue necrosis factor-α (TNF-α) in serum. DCF dose dependently induced ulcers that were associated with both a significant (P < 0.05) loss of Hb and an increase in the level of MPO and TNF-α, with severity of ulceration highest at 18 hours. Pretreatment with TPPU dose dependently prevented ulcer formation by DCF, increased the levels of epoxy fatty acids, including EETs, and TPPU's efficacy was comparable to OME. TPPU significantly (P < 0.05) reversed the effect of DCF on the level of Hb, MPO, and TNF-α Thus sEHI might be useful in the management of NSAID-induced ulcers.

Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE2 was monitored. While OME treatment by itself exhibited variable effects on PGE2 induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study.

Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl4)-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl4-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl4-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl4-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl4, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress.

Dr. Bernard Brodie’s legacy is built on fundamental discoveries in pharmacology and drug metabolism that were then translated to the clinic to improve patient care. Similarly, the development of a novel class of therapeutics termed the soluble epoxide hydrolase (sEH) inhibitors was originally spurred by fundamental research exploring the biochemistry and physiology of the sEH. Here, we present an overview of the history and current state of research on epoxide hydrolases, specifically focusing on sEHs. In doing so, we start with the translational project studying the metabolism of the insect juvenile hormone mimic R-20458 [(E)-6,7-epoxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene], which led to the identification of the mammalian sEH. Further investigation of this enzyme and its substrates, including the epoxyeicosatrienoic acids, led to insight into mechanisms of inflammation, chronic and neuropathic pain, angiogenesis, and other physiologic processes. This basic knowledge in turn led to the development of potent inhibitors of the sEH that are promising therapeutics for pain, hypertension, chronic obstructive pulmonary disorder, arthritis, and other disorders.

Epoxy FAs (EpFAs) are important lipid mediators that are mainly metabolized by soluble epoxide hydrolase (sEH). Thus, sEH inhibition is a promising therapeutic target to treat numerous ailments. Several sEH polymorphisms result in amino acid substitutions and alter enzyme activity. K55R and R287Q are associated with inflammatory, cardiovascular, and metabolic diseases. R287Q seems to affect sEH activity through reducing formation of a catalytically active dimer. Thus, understanding how these SNPs affect the selectivity of sEH for substrates and inhibitors is of potential clinical importance. We investigated the selectivity of four sEH SNPs toward a series of EpFAs and inhibitors. We found that the SNPs alter the catalytic activity of the enzyme but do not alter the relative substrate and inhibitor selectivity. We also determined their dimer/monomer constants (KD/M). The WT sEH formed a very tight dimer, with a KD/M in the low picomolar range. Only R287Q resulted in a large change of the KD/M. However, human tissue concentrations of sEH suggest that it is always in its dimer form independently of the SNP. These results suggest that the different biologies associated with K55R and R287Q are not explained by alteration in dimer formation or substrate selectivity.

Epoxygenated fatty acids (EpFAs), which are lipid mediators produced by cytochrome P450 epoxygenases from polyunsaturated fatty acids, are important signaling molecules known to regulate various biological processes including inflammation, pain and angiogenesis. The EpFAs are further metabolized by soluble epoxide hydrolase (sEH) to form fatty acid diols which are usually less-active. Pharmacological inhibitors of sEH that stabilize endogenous EpFAs are being considered for human clinical uses. Here we review the biology of ω-3 and ω-6 EpFAs on inflammation, pain, angiogenesis and tumorigenesis.

BackgroundUrban particulate matter (PM) has been epidemiologically correlated with multiple cardiopulmonary morbidities and mortalities, in sensitive populations. Children exposed to PM are more likely to develop respiratory infections and asthma. Although PM originates from natural and anthropogenic sources, vehicle exhaust rich in polycyclic aromatic hydrocarbons (PAH) can be a dominant contributor to the PM2.5and PM0.1fractions and has been implicated in the generation of reactive oxygen species (ROS).ObjectivesCurrent studies of ambient PM are confounded by the variable nature of PM, so we utilized a previously characterized ethylene-combusted premixed flame particles (PFP) with consistent and reproducible physiochemical properties and 1) measured the oxidative potential of PFP compared to ambient PM, 2) determined the ability of PFPs to generate oxidative stress and activate the transcription factor usingin vitroandex vivomodels, and 3) we correlated these responses with antioxidant enzyme expressionin vivo.MethodsWe compared oxidative stress response (HMOX1) and antioxidant enzyme (SOD1, SOD2, CAT, and PRDX6) expressionin vivoby performing a time-course study in 7-day old neonatal and young adult rats exposed to a single 6-hour exposure to 22.4 μg/m3PFPs.ResultsWe showed that PFP is a potent ROS generator that induces oxidative stress and activates Nrf2. Induction of the oxidative stress responsive enzyme HMOX1in vitrowas mediated through Nrf2 activation and was variably upregulated in both ages. Furthermore, antioxidant enzyme expression had age and lung compartment variations post exposure. Of particular interest was SOD1, which had mRNA and protein upregulation in adult parenchyma, but lacked a similar response in neonates.ConclusionsWe conclude that PFPs are effective ROS generators, comparable to urban ambient PM2.5,that induce oxidative stress in neonatal and adult rat lungs. PFPs upregulate a select set of antioxidant enzymes in young adult animals, that are unaffected in neonates. We conclude that the inability of neonatal animals to upregulate the antioxidant response may, in part, explain enhanced their susceptibility to ultrafine particles, such as PFP.

Vehicle exhaust is rich in polycyclic aromatic hydrocarbons (PAHs) and is a dominant contributor to urban particulate pollution (PM). Exposure to PM is linked to respiratory and cardiovascular morbidity and mortality in susceptible populations, such as children. PM can contribute to the development and exacerbation of asthma, and this is thought to occur because of the presence of electrophiles in PM or through electrophile generation via the metabolism of PAHs. Glutathione (GSH), an abundant intracellular antioxidant, confers cytoprotection through conjugation of electrophiles and reduction of reactive oxygen species. GSH-dependent phase II detoxifying enzymes glutathione peroxidase and glutathione S-transferase facilitate metabolism and conjugation, respectively. Ambient particulates are highly variable in composition, which complicates systematic study. In response, we have developed a replicable ultrafine premixed flame particle (PFP)-generating system for in vivo studies. To determine particle effects in the developing lung, 7-day-old neonatal and adult rats inhaled 22 μg/m(3) PFP during a single 6-hour exposure. Pulmonary GSH and related phase II detoxifying gene and protein expression were evaluated 2, 24, and 48 hours after exposure. Neonates exhibited significant depletion of GSH despite higher initial baseline levels of GSH. Furthermore, we observed attenuated induction of phase II enzymes (glutamate cysteine ligase, glutathione reductase, glutathione S-transferase, and glutathione peroxidase) in neonates compared with adult rats. We conclude that developing neonates have a limited ability to deviate from their normal developmental pattern that precludes adequate adaptation to environmental pollutants, which results in enhanced cytotoxicity from inhaled PM.