sEH-derived metabolites of linoleic acid drive pathologic inflammation while impairing key innate immune cell function in burn injury.

Fatty acid composition in the Western diet has shifted from saturated to polyunsaturated fatty acids (PUFAs), and specifically to linoleic acid (LA, 18:2), which has gradually increased in the diet over the past 50 y to become the most abundant dietary fatty acid in human adipose tissue. PUFA-derived oxylipins regulate a variety of biological functions. The cytochrome P450 (CYP450)­formed epoxy fatty acid metabolites of LA (EpOMEs) are hydrolyzed by the soluble epoxide hydrolase enzyme (sEH) to dihydroxyoctadecenoic acids (DiHOMEs). DiHOMEs are considered cardioprotective at low concentrations but at higher levels have been implicated as vascular permeability and cytotoxic agents and are associated with acute respiratory distress syndrome in severe COVID-19 patients. High EpOME levels have also correlated with sepsis-related fatalities; however, those studies failed to monitor DiHOME levels. Considering the overlap of burn pathophysiology with these pathologies, the role of DiHOMEs in the immune response to burn injury was investigated. 12,13-DiHOME was found to facilitate the maturation and activation of stimulated neutrophils, while impeding monocyte and macrophage functionality and cytokine generation. In addition, DiHOME serum concentrations were significantly elevated in burn-injured mice and these increases were ablated by administration of 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a sEH inhibitor. TPPU also reduced necrosis of innate and adaptive immune cells in burned mice, in a dose-dependent manner. The findings suggest DiHOMEs are a key driver of immune cell dysfunction in severe burn injury through hyperinflammatory neutrophilic and impaired monocytic actions, and inhibition of sEH might be a promising therapeutic strategy to mitigate deleterious outcomes in burn patients.

Soluble epoxide hydrolase is an endogenous regulator of obesity-induced intestinal barrier dysfunction and bacterial translocation.

Intestinal barrier dysfunction, which leads to translocation of bacteria or toxic bacterial products from the gut into bloodstream and results in systemic inflammation, is a key pathogenic factor in many human diseases. However, the molecular mechanisms leading to intestinal barrier defects are not well understood, and there are currently no available therapeutic approaches to target intestinal barrier function. Here we show that soluble epoxide hydrolase (sEH) is an endogenous regulator of obesity-induced intestinal barrier dysfunction. We find that sEH is overexpressed in the colons of obese mice. In addition, pharmacologic inhibition or genetic ablation of sEH abolishes obesity-induced gut leakage, translocation of endotoxin lipopolysaccharide or bacteria, and bacterial invasion-induced adipose inflammation. Furthermore, systematic treatment with sEH-produced lipid metabolites, dihydroxyeicosatrienoic acids, induces bacterial translocation and colonic inflammation in mice. The actions of sEH are mediated by gut bacteria-dependent mechanisms, since inhibition or genetic ablation of sEH fails to attenuate obesity-induced gut leakage and adipose inflammation in mice lacking gut bacteria. Overall, these results support that sEH is a potential therapeutic target for obesity-induced intestinal barrier dysfunction, and that sEH inhibitors, which have been evaluated in human clinical trials targeting other human disorders, could be promising agents for prevention and/or treatment.

Nanobodies for Accurate Recognition of Iso-tenuazonic Acid and Development of Sensitive Immunoassay for Contaminant Detection in Foods.

The accurate analysis of chemical isomers plays an important role in the study of their different toxic effects and targeted detection of pollutant isomers in foods. The Alternaria mycotoxins tenuazonic acid (TeA) and iso-tenuazonic acid (ITeA) are two isomer mycotoxins with the lack of single analysis methods due to the similar structures. Antibody-based immunoassays exhibit high sensitivity and superior application in isomer-specific determination. Previously, various kinds of antibodies for TeA have been prepared in our group. Herein, highly specific nanobodies (Nbs) against ITeA mycotoxin were selected from immune nanobody phage display library, and one of Nbs, namely Nb(B3G3) exhibited excellent affinity, thermal stability as well as organic solvent tolerance. By molecular simulation and docking technology, it was found that stronger interaction between Nb(B3G3) and ITeA lead to higher affinity than that for its isomer TeA. Furthermore, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was established with a limit of detection (LOD) of 0.09 ng/mL for ITeA mycotoxin. The recovery rate of ITeA in spiked samples was analyzed with 84.8%-89.5% for rice, 78.3%-96.3% for flour, and 79.5%-90.7% for bread. A conventional LC-MS/MS method was used to evaluate the accuracy of this proposed icELISA, which showed a satisfactory consistent correlation. Since the convenient strategy for nanobody generation by phage display technology, this study provide new biorecognition elements and sensitive immunoassay for analysis of ITeA in foods.

A Fast and Selective Approach for Profiling Vicinal Diols Using Liquid Chromatography-Post Column Derivatization-Double Precursor Ion Scanning Mass Spectrometry.

Vicinal diols are important signaling metabolites of various inflammatory diseases, and some of them are potential biomarkers for some diseases. Utilizing the rapid reaction between diol and 6-bromo-3-pyridinylboronic acid (BPBA), a selective and sensitive approach was established to profile these vicinal diols using liquid chromatography-post column derivatization coupled with double precursor ion scan-mass spectrometry (LC-PCD-DPIS-MS). After derivatization, all BPBA-vicinal-diol esters gave a pair of characteristic isotope ions resulting from 79Br and 81Br. The unique isotope pattern generated two characteristic fragment ions of m/z 200 and 202. Compared to a traditional offline derivatization technique, the new LC-PCD-DPIS-MS method retained the capacity of LC separation. In addition, it is more sensitive and selective than a full scan MS method. As an application, an in vitro study of the metabolism of epoxy fatty acids by human soluble epoxide hydrolase was tested. These vicinal-diol metabolites of individual regioisomers from different types of polyunsaturated fatty acids were easily identified. The limit of detection (LOD) reached as low as 25 nM. The newly developed LC-PCD-DPIS-MS method shows significant advantages in improving the selectivity and therefore can be employed as a powerful tool for profiling vicinal-diol compounds from biological matrices.

Lipidomic profiling reveals soluble epoxide hydrolase as a therapeutic target of obesity-induced colonic inflammation.

Obesity is associated with enhanced colonic inflammation, which is a major risk factor for colorectal cancer. Considering the obesity epidemic in Western countries, it is important to identify novel therapeutic targets for obesity-induced colonic inflammation, to develop targeted strategies for prevention. Eicosanoids are endogenous lipid signaling molecules involved in regulating inflammation and immune responses. Using an LC-MS/MS-based lipidomics approach, we find that obesity-induced colonic inflammation is associated with increased expression of soluble epoxide hydrolase (sEH) and its eicosanoid metabolites, termed fatty acid diols, in colon tissue. Furthermore, we find that pharmacological inhibition or genetic ablation of sEH reduces colonic concentrations of fatty acid diols, attenuates obesity-induced colonic inflammation, and decreases obesity-induced activation of Wnt signaling in mice. Together, these results support that sEH could be a novel therapeutic target for obesity-induced colonic inflammation and associated diseases.

A Soluble Epoxide Hydrolase Inhibitor, 1-TrifluoromethoxyPhenyl-3-(1-Propionylpiperidin-4-yl) Urea, Ameliorates Experimental Autoimmune Encephalomyelitis.

Polyunsaturated fatty acids (PUFAs) are essential FAs for human health. Cytochrome P450 oxygenates PUFAs to produce anti-inflammatory and pain-resolving epoxy fatty acids (EpFAs) and other oxylipins whose epoxide ring is opened by the soluble epoxide hydrolase (sEH/Ephx2), resulting in the formation of toxic and pro-inflammatory vicinal diols (dihydroxy-FAs). Pharmacological inhibition of sEH is a promising strategy for the treatment of pain, inflammation, cardiovascular diseases, and other conditions. We tested the efficacy of a potent, selective sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Prophylactic TPPU treatment significantly ameliorated EAE without affecting circulating white blood cell counts. TPPU accumulated in the spinal cords (SCs), which was correlated with plasma TPPU concentration. Targeted lipidomics in EAE SCs and plasma identified that TPPU blocked production of dihydroxy-FAs efficiently and increased some EpFA species including 12(13)-epoxy-octadecenoic acid (12(13)-EpOME) and 17(18)-epoxy-eicosatrienoic acid (17(18)-EpETE). TPPU did not alter levels of cyclooxygenase (COX-1/2) metabolites, while it increased 12-hydroxyeicosatetraenoic acid (12-HETE) and other 12/15-lipoxygenase metabolites. These analytical results are consistent with sEH inhibitors that reduce neuroinflammation and accelerate anti-inflammatory responses, providing the possibility that sEH inhibitors could be used as a disease modifying therapy, as well as for MS-associated pain relief.

An Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Heptamer Fusion for the Detection of Tetrabromobisphenol A in Sediment.

Tetrabromobisphenol A (TBBPA) is a flame retardant and has become a widely concerning environmental pollutant. An ultrasensitive nanobody-based immunoassay was developed to monitor the exposure of TBBPA in sediment. First, the anti-TBBPA nanobody was fused with nanoluciferase, and then a one-step bioluminescent enzyme immunoassay (BLEIA) was developed with high sensitivity for TBBPA, with a maximum half inhibition concentration (IC50) at 187 pg/mL. Although approximately 10-fold higher sensitivity can be achieved by this developed BLEIA than by the classical two-step ELISA (IC50 at 1778 pg/mL), it is still a challenge to detect trace TBBPA in sediment samples reliably due to the relatively high matrix effect. To further improve the performance of this one-step BLEIA, a C4b-binding protein (C4BP) was inserted as a self-assembling linker between the nanobody and nanoluciferase. Therefore, a heptamer fusion containing seven binders and seven tracers was generated. This reagent improved the binding capacity and signal amplification. The one-step heptamer plus BLEIA based on this immune-reagent shows an additional 7-fold improvement of sensitivity, with the IC50 of 28.9 pg/mL and the limit of detection as low as 2.5 pg/mL. The proposed assay was further applied to determine the trace TBBPA in sediment, and the recovery was within 92-103%. Taking advantage of this heptamer fusion, one-step BLEIA can serve as a powerful tool for fast detection of trace TBBPA in the sediment samples.

Chemiluminescent Enzyme Immunoassay and Bioluminescent Enzyme Immunoassay for Tenuazonic Acid Mycotoxin by Exploitation of Nanobody and Nanobody-Nanoluciferase Fusion.

The isolation of nanobodies (Nbs) from phage display libraries is an increasingly effective approach for the generation of new biorecognition elements, which can be used to develop immunoassays. In this study, highly specific Nbs against the Alternaria mycotoxin tenuazonic acid (TeA) were isolated from an immune nanobody phage display library using a stringent biopanning strategy. The obtained Nbs were characterized by classical enzyme-linked immunosorbent assay (ELISA), and the best one Nb-3F9 was fused with nanoluciferase to prepare an advanced bifunctional fusion named nanobody-nanoluciferase (Nb-Nluc). In order to improve the sensitivity and reduce the assay time, two different kinds of luminescent strategies including chemiluminescent enzyme immunoassay (CLEIA) and bioluminescent enzyme immunoassay (BLEIA) were established, respectively, on the basis of the single Nb and the fusion protein Nb-Nluc for TeA detection. The two-step CLEIA was developed on the basis of the same nanobody as ELISA, only with simple substrate replacement from 3,3',5,5'-tetramethylbenzidine (TMB) to luminol. In contrast with CLEIA, the novel BLEIA was conducted in one-step new strategy on the basis of Nb-Nluc and bioluminescent substrate coelenterazine-h (CTZ-h). Their half maximal inhibitory concentration (IC50) values were similar to 8.6 ng/mL for CLEIA and 9.3 ng/mL for BLEIA, which was a 6-fold improvement in sensitivity compared with that of ELISA (IC50 of 54.8 ng/mL). Both of the two assays provided satisfactory recoveries ranging from 80.1%-113.5% in real samples, which showed better selectivity for TeA analogues and other common mycotoxins. These results suggested that Nbs and Nb-Nluc could be used as useful reagents for immunodetection and that the developed CLEIA/BLEIA have great potential for TeA analysis.

N-Benzyl-linoleamide, a Constituent of Lepidium meyenii (Maca), Is an Orally Bioavailable Soluble Epoxide Hydrolase Inhibitor That Alleviates Inflammatory Pain.

Lepidium meyenii (maca), a plant indigenous to the Peruvian Andes, recently has been utilized globally for claimed health or recreational benefits. The search for natural products that inhibit soluble epoxide hydrolase (sEH), with therapeutically relevant potencies and concentrations, led to the present study on bioactive amide secondary metabolites found in L. meyenii, the macamides. Based on known and suspected macamides, 19 possible macamides were synthesized and characterized. The majority of these amides displayed excellent inhibitory potency (IC50 ≈ 20-300 nM) toward the recombinant mouse, rat, and human sEH. Quantitative analysis of commercial maca products revealed that certain products contain known macamides (1-5, 8-12) at therapeutically relevant total concentrations (≥3.29 mg/g of root), while the inhibitory potency of L. meyenii extracts directly correlates with the sum of concentration/IC50 ratios of macamides present. Considering both its in vitro efficacy and high abundance in commercial products, N-benzyl-linoleamide (4) was identified as a particularly relevant macamide that can be utilized for in vivo studies. Following oral administration in the rat, compound 4 not only displayed acceptable pharmacokinetic characteristics but effectively reduced lipopolysaccharide-induced inflammatory pain. Inhibition of sEH by macamides provides a plausible biological mechanism of action to account for several beneficial effects previously observed with L. meyenii treatments.

Novel metabolomic method to assess the effect-based removal efficiency of advanced wastewater treatment techniques.

Unprecedented levels of chemicals of anthropogenic origin are currently released into surface waters globally. Wastewater treatment plant effluent has been identified as a major source, containing a broad mixture of pharmaceuticals and consumer chemicals. Therefore, there is a need for implementation of advanced wastewater treatment techniques, such as ozonation and adsorption methods, to reduce the contamination. However, there are conflicting findings on the toxicity of treated effluent and only limited possibilities for assessing the effect-based removal efficiency (EBRE) of different treatment techniques. Here, we describe a metabolomics approach to detect perturbations in fatty acid catabolic pathways as a proxy for biological effects. Metabolites in three fatty acid pathways were analyzed in a common damselfly larva (Coenagrion hastulatum) by liquid chromatography coupled to mass spectrometry. The larvae were exposed for one week to either conventionally treated effluent (activated sludge treatment), effluent additionally treated with ozone or effluent additionally treated with biochar filtration and results were compared with those from tap water control exposure. Five lipoxygenase-derived oxylipins (9,10,13-TriHOME, 9,12,13-TriHOME, 9-HODE, 9-HOTrE, and 13-HOTrE) decreased in response to conventionally treated effluent exposure. By using an additional treatment step, oxylipin levels were restored with exception of 9,10,13-TriHOME (ozonated effluent), and 9-HOTrE and 13-HOTrE (effluent filtered with biochar). In conclusion, exposure to wastewater effluent affected fatty acid metabolite levels in damselfly larvae, and a subset of the analyzed metabolites may serve as indicators for biological effects in biota in response to effluent exposure. To that effect, our findings suggest a new metabolomics protocol for assessing EBRE.

Soluble Epoxide Hydrolase Inhibition by t-TUCB Promotes Brown Adipogenesis and Reduces Serum Triglycerides in Diet-Induced Obesity.

Brown adipose tissue (BAT) is an important target for obesity treatment and prevention. Soluble epoxide hydrolase (sEH) converts bioactive epoxy fatty acids (EpFAs) into less active diols. sEH inhibitors (sEHI) are beneficial in many chronic diseases by stabilizing EpFAs. However, roles of sEH and sEHI in brown adipogenesis and BAT activity in treating diet-induced obesity (DIO) have not been reported. sEH expression was studied in in vitro models of brown adipogenesis and the fat tissues of DIO mice. The effects of the sEHI, trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy-benzoic acid (t-TUCB), were studied in vitro and in the obese mice via mini osmotic pump delivery. sEH expression was increased in brown adipogenesis and the BAT of the DIO mice. t-TUCB promoted brown adipogenesis in vitro. Although t-TCUB did not change body weight, fat pad weight, or glucose and insulin tolerance in the obese mice, it decreased serum triglycerides and increased protein expression of genes important for lipid metabolism in the BAT. Our results suggest that sEH may play a critical role in brown adipogenesis, and sEHI may be beneficial in improving BAT protein expression involved in lipid metabolism. Further studies using the sEHI combined with EpFA generating diets for obesity treatment and prevention are warranted.

Quantitative Detection of Fipronil and Fipronil-Sulfone in Sera of Black-Tailed Prairie Dogs and Rats after Oral Exposure to Fipronil by Camel Single-Domain Antibody-Based Immunoassays.

The insecticide fipronil can be metabolized to its sulfone in mammalian species. Two camel single-domain antibodies (VHHs) F1 and F6, selective to fipronil and fipronil-sulfone, respectively, were generated and used to develop enzyme linked immunosorbent assays (ELISAs) for the detection of the two compounds in the sera of black-tailed prairie dogs and rats. The limits of detection of fipronil and fipronil-sulfone in the rodent sera by the corresponding ELISAs were 10 and 30 ng mL-1, and the linear ranges were 30-1000 and 75-2200 ng mL-1. ELISAs showed a good recovery for fipronil and fipronil-sulfone cospiked in the control sera of the black-tailed prairie dogs (90-109%) and rats (93-106%). The VHH-based ELISAs detected fipronil and fipronil-sulfone in the sera of the rodents that received a repeated oral administration of fipronil. The average concentration of fipronil-sulfone was approximately 3.2-fold higher than fipronil in the prairie dog sera (1.15 vs 0.36 µg mL-1) and rat sera (1.77 vs 0.53 µg mL-1). ELISAs agreed well with a liquid chromatography-mass spectrometry method for the quantification of both fipronil and fipronil-sulfone in real serum samples. Fipronil-sulfone was identified as the predominant metabolite of fipronil in the black-tailed prairie dog and rat sera.

Lipidomes of brain from rats acutely intoxicated with diisopropylfluorophosphate identifies potential therapeutic targets.

Organophosphates (OPs), a class of phosphorus-containing chemicals that act by disrupting cholinergic transmission, include both toxic and fast-acting chemical warfare agents as well as less toxic but more easily accessible OP pesticides. The classical atropine/2-PAM antidote fails to protect against long-term symptoms following acute intoxication with OPs at levels that trigger status epilepticus. Acute OP intoxication also causes a robust neuroinflammatory response, which is implicated in the pathogenesis of long-term effects. In this study, we characterized the profiles of lipid mediators, important players in neuroinflammation, in the rat model of acute DFP intoxication. The profiles of lipid mediators were monitored in three different regions of the brain (cortex, hippocampus, and cerebellum) at 0, 1, 3, 7, 14, and 28 days post-exposure. The distribution pattern of lipid mediators was distinct in the three brain regions. In the cerebellum, the profile is dominated by LOX metabolites, while the lipid mediator profiles in cortex and hippocampus are dominated by COX metabolites followed by LOX and CYP 450 metabolites. Following acute DFP intoxication, most of the pro-inflammatory lipid mediators (e.g., PGD2 and PGE2) increased rapidly from day 1, while the concentrations of some anti-inflammatory lipid mediators (e.g. 14,15 EpETrE) decreased after DFP intoxication but recovered by day 14 post-exposure. The lipidomics results suggest two potential treatment targets: blocking the formation of prostaglandins by inhibiting COX and stabilizing the anti-inflammatory lipid mediators containing epoxides by inhibiting the enzyme soluble epoxide hydrolase (sEH).

A new sensitive LC/MS/MS analysis of vitamin D metabolites using a click derivatization reagent, 2-nitrosopyridine.

There is an increased demand for comprehensive analysis of vitamin D metabolites. This is a major challenge, especially for 1α,25-dihydroxyvitamin D [1α,25(OH)2VitD], because it is biologically active at picomolar concentrations. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) was a revolutionary reagent in dramatically increasing sensitivity of all diene metabolites and allowing the routine analysis of the bioactive, but minor, vitamin D metabolites. A second generation of reagents used large fixed charge groups that increased sensitivity at the cost of a deterioration in chromatographic separation of the vitamin D derivatives. This precludes a survey of numerous vitamin D metabolites without redesigning the chromatographic system used. 2-Nitrosopyridine (PyrNO) demonstrates that one can improve ionization and gain higher sensitivity over PTAD. The resulting vitamin D derivatives facilitate high-resolution chromatographic separation of the major metabolites. Additionally, a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE) was developed to selectively extract 1α,25(OH)2VitD, while reducing 2- to 4-fold ion suppression compared with SPE alone. LLE-SPE followed by PyrNO derivatization and LC/MS/MS analysis is a promising new method for quantifying vitamin D metabolites in a smaller sample volume (100 µL of serum) than previously reported methods. The PyrNO derivatization method is based on the Diels-Alder reaction and thus is generally applicable to a variety diene analytes.

High-throughput screening of triplex DNA binders from complicated samples by 96-well pate format in conjunction with peak area-fading UHPLC-Orbitrap MS.

Conventional strategies for the screening of DNA triplex binders cannot be used for complicated samples, such as ligand libraries created by combinatorial chemistry or from natural product extracts. In the current study, an ultra-high-performance liquid chromatography coupled with an Orbitrap mass spectrometry (UHPLC-Orbitrap-MS)-based approach, which we call peak area-fading (PAF) UHPLC-Orbitrap-MS and was designed for just such a purpose, is reported. The triplex DNA modified 96-well plate and the single stranded oligonucleotide modified 96-well plate (as control) were incubated with ligand libraries, and the unbound ligands were directly determined via UHPLC-ESI-MS. The binders were detected through the decrease (fading) in the peak areas compared to those of the control group. Several factors, such as incubation time, incubation temperature, and buffer, which might affect the binding affinity and reproducibility, were optimized. The potential of the approach was examined using the extracts of Rhizoma Coptidis and Phellodendron chinense Schneid cortexe. The triplex DNA-binding capabilities of the five components (epiberberine, coptisine, jatrorrhizine, berberrubine, and columbamine) were found for the first time, indicating their efficiency for the analysis of complicated samples. In contrast to our previous study, which suffered from a serious drawback of poor reproducibility, this method is more robust and more suitable for high-throughput measurements, opening a new experimental strategy in assessing large libraries of potential drug candidates that work by forming a drug/DNA complex.

Anti-Ulcer Efficacy of Soluble Epoxide Hydrolase Inhibitor TPPU on Diclofenac-Induced Intestinal Ulcers.

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.

Catalytic Activities of Tumor-Specific Human Cytochrome P450 CYP2W1 Toward Endogenous Substrates.

CYP2W1 is a recently discovered human cytochrome P450 enzyme with a distinctive tumor-specific expression pattern. We show here that CYP2W1 exhibits tight binding affinities for retinoids, which have low nanomolar binding constants, and much poorer binding constants in the micromolar range for four other ligands. CYP2W1 converts all-transretinoic acid (atRA) to 4-hydroxyatRA and all-transretinol to 4-OH all-transretinol, and it also oxidizes retinal. The enzyme much less efficiently oxidizes 17ß-estradiol to 2-hydroxy-(17ß)-estradiol and farnesol to a monohydroxylated product; arachidonic acid is, at best, a negligible substrate. These findings indicate that CYP2W1 probably plays an important role in localized retinoid metabolism that may be intimately linked to its involvement in tumor development.

Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal.

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28-AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06-0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.

Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE2 induced pain model.

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 (100mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3mg/kg/day, p.o.) and OME (100mg/kg/day, p.o., 7 days)+TPPU (3mg/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.

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase.

The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer, and other diseases. However, there is not a simple, inexpensive, and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage-displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the enzyme-linked immunosorbent assay (ELISA) and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney, and lung). The VHH-based ELISA appears to be a new reliable method for monitoring the sEH and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research.