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Inhibition of Microsomal Epoxide Hydrolases by Ureas, Amides, and Amines
Abstract
The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to several diseases, such as emphysema, spontaneous abortion, and several forms of cancer. To provide new tools for studying the function of mEH, inhibition of this enzyme was investigated. Inhibition of recombinant rat and human mEH was achieved using primary ureas, amides, and amines. Several of these compounds are more potent than previously published inhibitors. Elaidamide, the most potent inhibitor that is obtained, has a Ki of 70 nM for recombinant rat mEH. This compound interacts with the enzyme forming a noncovalent complex, and blocks substrate turnover through an apparent mix of competitive and noncompetitive inhibition kinetics. Furthermore, in insect cell cultures expressing rat mEH, elaidamide enhances the toxicity effects of epoxide-containing xenobiotics. These inhibitors could be valuable tools for investigating the physiological and toxicological roles of mEH.
... The following Cyp inhibitors were purchased from Sigma-Aldrich: furafylline (Cyp1a2), α-naphthoflavone (Cyp1b1), tranylcypromine (Cyp2c29, Cyp2c39), quercetin (Cyp2c29), quinidine (Cyp2d9), diethyldithiocarbamate (Cyp2e1) and ketoconazole (Cyp3a11). The specific epoxide hydrolase inhibitor elaidamide was synthesized and kindly provided by Dr Christophe Morisseau [30]. Aroclor 1254 was obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). ...
... Epoxide hydrolase inhibition. Because the 3',4'-dihydrodiol-FAA was likely generated from the hydolysis of the 3',4'-epoxy-FAA by the epoxide hydrolase, we determined the role of this enzyme using elaidamide, an epoxide hydrolase inhibitor [30], at final concentrations of 2, 4 and 10 µM. Evaluation of direct or indirect hydroxylation. ...
... In presence of NAC, the 4'-OH-FAA, the 3'-OH-FAA and the 6-OH-FAA, were significantly reduced, indicating that a significant percentage of these hydroxylated metabolites were indeed derived from the non enzymatic isomerization of their corresponding epoxides. The involvement of epoxide hydrolase in the formation of the 3',4'dihydrodiol-FAA was also confirmed using elaidamide, a specific inhibitor of this enzyme [30]. ...
The metabolism of flavone-8-acetic acid (FAA) has been hypothesized to be partly responsible for its potent anticancer activity in mice. The purpose of this study was to identify the mouse enzymes involved in FAA Phase I metabolism and evaluate their possible induction in vivo by FAA. Mouse microsomes metabolized FAA into 6 metabolites: 3',4'-dihydrodiol-FAA, 5,6-epoxy-FAA, 4'-OH-FAA, 3'-OH-FAA, 3',4'-epoxy-FAA and 6-OH-FAA. Using Cyp-specific inhibitors (furafylline, Cyp1a2; α-naphthoflavone, Cyp1b1; tranylcypromine, Cyp2b9; quercetin, Cyp2c29; quinidine, 2d9; diethyldithiocarbamate, Cyp2e1; ketoconazole, Cyp3a11), the formation of 5,6-epoxy-FAA was mainly attributed to Cyps 1a2, 1b1, 2b9, 2c29 and 2e1, whereas the 3',4'-epoxy-FAA was formed by Cyps 2b9 and 3a11. The 4'-OH-FAA was generated by Cyps 1a2, 1b1, 2b9 and 2e1, and the 6-OH-FAA was formed by Cyps 1b1 and 2c9. Using the epoxide scavenger N-acetyl cysteine, 4'-OH-FAA, 3'-OH-FAA and 6-OH-FAA were shown to derive partly from non enzymatic isomerisation of their corresponding epoxides. The specific epoxide hydrolase inhibitor elaidamide allowed the confirmation that 3',4'-dihydrodiol-FAA was formed via the epoxide hydrolase. FAA treatment in vivo in mice led to a significant increase in the hepatic expression of Cyp1a2 (1.9-fold), 2e1 (2.1-fold), 2b10 (3.2-fold), 2d9 (2.3-fold) and 3a11 (2.2-fold), as evaluated by qRT-PCR. In conclusion, several Cyps were shown to be involved in FAA metabolism, particularly Cyps 3a11 and 2b9 which were responsible for the formation of the principal metabolites (5,6-epoxy-FAA, 3',4'-epoxy-FAA), and that FAA could induce the expression of several Cyps after in vivo administration. The possible implication of these enzymes in the in vivo anticancer activity of FAA in mice is discussed.
... The following Cyp inhibitors were purchased from Sigma-Aldrich: furafylline (Cyp1a2), α-naphthoflavone (Cyp1b1), tranylcypromine (Cyp2c29, Cyp2c39), quercetin (Cyp2c29), quinidine (Cyp2d9), diethyldithiocarbamate (Cyp2e1) and ketoconazole (Cyp3a11). The specific epoxide hydrolase inhibitor elaidamide was synthesized and kindly provided by Dr Christophe Morisseau [30]. Aroclor 1254 was obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). ...
... Epoxide hydrolase inhibition. Because the 3',4'-dihydrodiol-FAA was likely generated from the hydolysis of the 3',4'-epoxy-FAA by the epoxide hydrolase, we determined the role of this enzyme using elaidamide, an epoxide hydrolase inhibitor [30], at final concentrations of 2, 4 and 10 µM. Evaluation of direct or indirect hydroxylation. ...
... In presence of NAC, the 4'-OH-FAA, the 3'-OH-FAA and the 6-OH-FAA, were significantly reduced, indicating that a significant percentage of these hydroxylated metabolites were indeed derived from the non enzymatic isomerization of their corresponding epoxides. The involvement of epoxide hydrolase in the formation of the 3',4'dihydrodiol-FAA was also confirmed using elaidamide, a specific inhibitor of this enzyme [30]. ...
The metabolism of flavone-8-acetic acid (FAA) has been hypothesized to be partly responsible for its potent anticancer activity in mice. The purpose of this study was to identify the mouse enzymes involved in FAA Phase I metabolism and evaluate their possible induction in vivo by FAA. Mouse microsomes metabolized FAA into 6 metabolites: 3',4'-dihydrodiol-FAA, 5,6-epoxy-FAA, 4'-OH-FAA, 3'-OH-FAA, 3',4'-epoxy-FAA and 6-OH-FAA. Using Cyp-specific inhibitors (furafylline, Cyp1a2; α-naphthoflavone, Cyp1b1; tranylcypromine, Cyp2b9; quercetin, Cyp2c29; quinidine, 2d9; diethyldithiocarbamate, Cyp2e1; ketoconazole, Cyp3a11), the formation of 5,6-epoxy-FAA was mainly attributed to Cyps 1a2, 1b1, 2b9, 2c29 and 2e1, whereas the 3',4'-epoxy-FAA was formed by Cyps 2b9 and 3a11. The 4'-OH-FAA was generated by Cyps 1a2, 1b1, 2b9 and 2e1, and the 6-OH-FAA was formed by Cyps 1b1 and 2c9. Using the epoxide scavenger N-acetyl cysteine, 4'-OH-FAA, 3'-OH-FAA and 6-OH-FAA were shown to derive partly from non enzymatic isomerisation of their corresponding epoxides. The specific epoxide hydrolase inhibitor elaidamide allowed the confirmation that 3',4'-dihydrodiol-FAA was formed via the epoxide hydrolase. FAA treatment in vivo in mice led to a significant increase in the hepatic expression of Cyp1a2 (1.9-fold), 2e1 (2.1-fold), 2b10 (3.2-fold), 2d9 (2.3-fold) and 3a11 (2.2-fold), as evaluated by qRT-PCR. In conclusion, several Cyps were shown to be involved in FAA metabolism, particularly Cyps 3a11 and 2b9 which were responsible for the formation of the principal metabolites (5,6-epoxy-FAA, 3',4'-epoxy-FAA), and that FAA could induce the expression of several Cyps after in vivo administration. The possible implication of these enzymes in the in vivo anticancer activity of FAA in mice is discussed.
... Over the past decade, we have developed more potent mEH inhibitors to study the biological roles of mEH [32,33]. 0003-2697/$ -see front matter Ó Traditionally, tritium-labeled cis-stilbene oxide ([ 3 H]cSO) was used as substrate to measure inhibitor potency. ...
... The inhibition potency (IC 50 ) of three described mEH inhibitors [32,33] was measured with compound 11 as substrate and with [ 3 H]cSO as described previously [33] for comparison. In black 96well plates, to 170 ll of human mEH (2.0 lg/ml) or rat mEH (0.6 lg/ml) in Tris-HCl buffer (100 mM, pH 8.5) containing 0.1 mg/ml BSA, 2 ll of the inhibitors in DMSO solution was added. ...
... Over the past decade, we have developed more potent mEH inhibitors to study the biological roles of mEH [32,33]. ...
The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure-activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z' value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.
... However, since these compounds contain an epoxide, one might expect that they will be substrates of EH and glutathione Stransferase, thus inhibition may be transient. Recently Morisseau et al. (1999Morisseau et al. ( , 2001 reported amide, urea and carbamate inhibitors of mammalian soluble and microsomal epoxide hydrolases that are EH-stable and have IC 50 s in the low nanomolar and micromolar ranges, respectively. Because these compounds act by mimicking a transition state intermediate between the enzyme and the epoxide (Argiriadi et al., 1999Yamada et al., 2000), and because the overall mechanism of JHEH is similar to the mammalian EH (Debernard et al., 1998;Linderman et al., 2000), one could expect that compounds with similar pharmacophores will also inhibit JHEH. ...
... The general methods for making these compounds have been described in previous papers (Argiriadi et al., 1999;Morisseau et al., 1999Morisseau et al., , 2001. In most cases the compounds were prepared by the reaction of a primary amine (or alcohol) with an alkyl-or aryl-substituted isocyanate in solvents such as hexane, ether, acetone or dimethylformamide (DMF). ...
... While primary amines and amides were optimal inhibitors for the rat and human microsomal epoxide hydrolases (Morisseau et al., 2001), the JHEH of M. sexta was poorly inhibited by these compounds (Table 4). These results seem to indicate the presence of two hydrophobic pockets, one small and one relatively larger, on either side of the catalytic site of JHEH. ...
A new class of inhibitors of juvenile hormone epoxide hydrolase (JHEH) of Manduca sexta and further in vitro characterization of the enzyme are reported. The compounds are based on urea and amide pharmacophores that were previously demonstrated as effective inhibitors of mammalian soluble and microsomal epoxide hydrolases. The best inhibitors against JHEH activity so far within this class are N-[(Z)-9-octadecenyl]-N'-propylurea and N-hexadecyl-N'-propylurea, which inhibited hydrolysis of a surrogate substrate (t-DPPO) with an IC(50) around 90 nM. The importance of substitution number and type was investigated and results indicated that N, N'-disubstitution with asymmetric alkyl groups was favored. Potencies of pharmacophores decreased as follows: amide>urea>carbamate>carbodiimide>thiourea and thiocarbamate for N, N'-disubstituted compounds with symmetric substituents, and urea>amide>carbamate for compounds with asymmetric N, N'-substituents. JHEH hydrolyzes t-DPPO with a K(m) of 65.6 microM and a V(max) of 59 nmol min(-1) mg(-1) and has a substantially lower K(m) of 3.6 microM and higher V(max) of 322 nmol min(-1) mg(-1) for JH III. Although none of these compounds were potent inhibitors of hydrolysis of JH III by JHEH, they are the first leads toward inhibitors of JHEH that are not potentially subject to metabolism through epoxide degradation.
... Steady-state reactions with inhibitors were used to qualitatively identify enzyme classes that catalyze the thiazole bioactivation pathway. Inhibitors used were 1 μM elaidamide to inhibit microsomal epoxide hydrolase (Morisseau et al., 2001) and 1 mM 1-aminobenzotriazole (ABT) to target general cytochromes P450 (de Montellano, 2018). Individual inhibitor solutions were prepared in potassium phosphate buffer at pH 7.4 with < 0.1% methanol (final) as a co-solvent. ...
... The supernatants were separated from the protein pellet, dried, and resuspended in mobile phase (20% acetonitrile, 80% water + 0.1% formic acid) for HPLC analysis (Supporting Information, S5). For further validation of putative epoxides, sudoxicam and meloxicam HLM reactions were conducted with epoxide hydrolase inhibitor elaidamide (1 μM) (Morisseau et al., 2001) to facilitate epoxide accumulation and suppression of downstream alpha-dicarbonyl metabolites. ...
... The first reported mEH inhibitors were alternate substrates that were poorly turned over [7e9]. Previously, we showed that primary ureas, amides and amines can inhibit mEH with a single digit micromolar range potency [10]. Later, we developed stable inhibitors of the rat mEH with IC 50 in the high nanomolar range [11]. ...
... A range of phenylacetamides with either adamantylthio-, arylthio-or benzylthio-substituents at the a-carbon (10e17) were prepared and tested (Table 1). Adamantylthio- (10) and arylthio-substituted (11) phenylacetamides were, in general, poorer inhibitors of the human mEH compared to the benzylthioacetamides (12e17), probably due to steric factors. Compounds 16 and 17 were then obtained in pure form, and were five and twofold more effective, respectively, than the most potent inhibitors developed at that point (6,7). ...
Microsomal epoxide hydrolase (mEH) hydrolyzes a wide range of epoxide containing molecules. Although involved in the metabolism of xenobiotics, recent studies associate mEH with the onset and development of some diseases. This phenomenon is partially attributed to the significant role mEH plays in hydrolyzing endogenous lipid mediators, suggesting more complex and extensive physiological functions. In order to obtain pharmacological tools to further study the biology and therapeutic potential of this enzyme target, we describe the development of highly potent inhibitors of the human mEH with IC 50 values in the low nanomolar range, around 2 orders of magnitude more potent than previously obtained mEH inhibitors. Rationalization of binding through docking calculations of the inhibitors to a mEH homology model revealed that an acetamide, with a bulky aliphatic substituent at the α‐position, as well as an aryl ring 3–4 bonds away function as key pharmacophore units. No interaction was observed with the thioether moiety, suggesting it can be replaced by more metabolically stable (e.g. methylene) groups in future studies.
Support or Funding Information
National Institute of Health (NIH) R35 ES030443 and R01 GM076324‐11 grants; National Institute of Environmental Health Sciences (NIEHS) Superfund Research Program P42 ES004699 grant; National Science Foundation Awards 1827246, 1805510 and 1627539; Computation support from Rosetta Commons and the US National Science Foundation’s XSEDE program
Compound 62 : The most potent mEH inhibitor
Figure 1 Modeling of key molecular interactions in one binding mode. Left: interactions between amide group of 62 with D226, Y299 and Y374 side chain. Right: π–π stacking between 62 and W227.
Figure 2
... The pharmacophore of each compound is marked in red. (Kim et al., 2011) 1 Introduction of the amide group to obtain the metabolically stable MS-PPOH generated a product with high structural similarity to a certain class of mEH inhibitors (Morisseau et al., 2001) and to some extend to sEH inhibitors ( Figure 7). This fact was ignored so far but is addressed in a side project of this work. ...
... EETs, however, are rapidly converted by EHs to their usually less active vicinal diols (DHETs). MS-PPOH shows strong structural similarity to the amide class of mEH inhibitors (Morisseau et al., 2001). However, we could not observe any inhibitory effect on mEH at any MS-PPOH concentration used in our FIAs (Figure 32 A). ...
... The first reported mEH inhibitors were alternate substrates that were poorly turned over [7e9]. Previously, we showed that primary ureas, amides and amines can inhibit mEH with a single digit micromolar range potency [10]. Later, we developed stable inhibitors of the rat mEH with IC 50 in the high nanomolar range [11]. ...
... A range of phenylacetamides with either adamantylthio-, arylthio-or benzylthio-substituents at the a-carbon (10e17) were prepared and tested (Table 1). Adamantylthio- (10) and arylthio-substituted (11) phenylacetamides were, in general, poorer inhibitors of the human mEH compared to the benzylthioacetamides (12e17), probably due to steric factors. Compounds 16 and 17 were then obtained in pure form, and were five and twofold more effective, respectively, than the most potent inhibitors developed at that point (6,7). ...
Microsomal epoxide hydrolase (mEH) hydrolyzes a wide range of epoxide containing molecules. Although involved in the metabolism of xenobiotics, recent studies associate mEH with the onset and development of certain disease conditions. This phenomenon is partially attributed to the significant role mEH plays in hydrolyzing endogenous lipid mediators, suggesting more complex and extensive physiological functions. In order to obtain pharmacological tools to further study the biology and therapeutic potential of this enzyme target, we describe the development of highly potent 2-alkylthio acetamide inhibitors of the human mEH with IC50 values in the low nanomolar range. These are around 2 orders of magnitude more potent than previously obtained primary amine, amide and urea-based mEH inhibitors. Experimental assay results and rationalization of binding through docking calculations of inhibitors to a mEH homology model indicate that an amide connected to an alkyl side chain and a benzyl-thio function as key pharmacophore units.
... To exclude potentially contaminating sEH activities, murine liver microsomes were prepared from sEH-KO mice and the reactions with human liver microsomes were performed in the presence of the sEH inhibitor, TPPU (2 M). CHO was used to inhibit mEH activities (15,16); the final CHO concentrations were 250 M with murine or 800 M with human liver microsomes. Incubations containing 0.1% ethanol were performed as vehicle controls. ...
... Accordingly, we hypothesized that CYP-mEH interactions might also modulate the stereospecificity of microsomal metabolite formation. To test this hypothesis, we analyzed the NADPHdependent generation of AA-derived monoepoxides (EETs) by murine and human liver microsomes in the absence and presence of the mEH inhibitor, CHO (15,16). CHO was used in concentrations sufficient to inhibit microsomal DHET formation by more than 90% (data from achiral analysis, not shown). ...
A chiral lipidomics approach was established for comprehensive profiling of regio- and stereoisomeric monoepoxy and monohydroxy metabolites of long-chain PUFAs as generated enzymatically by cytochromes P450 (CYPs), lipoxygenases (LOXs), and cyclooxygenases (COXs) and, in part, also unspecific oxidations. The method relies on reversed-phase chiral-LC coupled with ESI/MS/MS. Applications revealed partially opposing enantioselectivities of soluble and microsomal epoxide hydrolases (mEHs). Ablation of the soluble epoxide hydrolase (sEH) gene resulted in specific alterations in the enantiomeric composition of endogenous monoepoxy metabolites. For example, the (R,S)/(S,R)-ratio of circulating 14,15-EET changed from 2.1:1 in WT to 9.7:1 in the sEH-KO mice. Studies with liver microsomes suggested that CYP/mEH interactions play a primary role in determining the enantiomeric composition of monoepoxy metabolites during their generation and release from the ER. Analysis of human plasma showed significant enantiomeric excess with several monoepoxy metabolites. Monohy-droxy metabolites were generally present as racemates; however, Ca²⁺-ionophore stimulation of whole blood samples resulted in enantioselective increases of LOX-derived metabolites (12S-HETE and 17S-hydroxydocosahexaenoic acid) and COX-derived metabolites (11R-HETE). Our chiral approach may provide novel opportunities for investigating the role of bioactive lipid mediators that generally exert their physiological functions in a highly regio- and stereospecific manner.—Blum, M., I. Dogan, M. Karber, M. Rothe, and W-H. Schunck. Chiral lipidomics of monoepoxy and monohydroxy metabolites derived from long-chain polyunsaturated fatty acids. J. Lipid Res. 2019. 60: 135–148. Copyright © 2019 Blum et al. Published under exclusive license by The American Society.
... Examining the chemical structure of valpromide showed that several amides are tightly bound mEH inhibitors with low nanomolar dissociation constants (Morisseau 2013). Consequently, a series of amide-based EH inhibitors were developed, and they are proven to be more potent than the epoxide-based mEH inhibitors (Morisseau et al. 2001). The mechanism of inhibition of amide-and urea-based EH inhibitors is by establishing hydrogen bonding between the carbonyl group of the inhibitor and tyrosine residues, as well as between the amino group of the inhibitor and aspartate residue, and therefore occupies the catalytic triad ( Fig. 2) (Morisseau 2013). ...
... The mechanism of inhibition of amide-and urea-based EH inhibitors is by establishing hydrogen bonding between the carbonyl group of the inhibitor and tyrosine residues, as well as between the amino group of the inhibitor and aspartate residue, and therefore occupies the catalytic triad ( Fig. 2) (Morisseau 2013). Their mEH inhibition kinetics is an apparent mix of competitive and non-competitive kinetics (Morisseau et al. 2001). An example for these inhibitors is elaidamide (Morisseau et al. 2008). ...
Epoxide hydrolases (EH) are ubiquitously expressed in all living organisms and in almost all organs and tissues. They are mainly subdivided into microsomal and soluble EH and catalyze the hydration of epoxides, three-membered-cyclic ethers, to their corresponding dihydrodiols. Owning to the high chemical reactivity of xenobiotic epoxides, microsomal EH is considered protective enzyme against mutagenic and carcinogenic initiation. Nevertheless, several endogenously produced epoxides of fatty acids function as important regulatory mediators. By mediating the formation of cytotoxic dihydrodiol fatty acids on the expense of cytoprotective epoxides of fatty acids, soluble EH is considered to have cytotoxic activity. Indeed, the attenuation of microsomal EH, achieved by chemical inhibitors or preexists due to specific genetic polymorphisms, is linked to the aggravation of the toxicity of xenobiotics, as well as the risk of cancer and inflammatory diseases, whereas soluble EH inhibition has been emerged as a promising intervention against several diseases, most importantly cardiovascular, lung and metabolic diseases. However, there is reportedly a significant overlap in substrate selectivity between microsomal and soluble EH. In addition, microsomal and soluble EH were found to have the same catalytic triad and identical molecular mechanism. Consequently, the physiological functions of microsomal and soluble EH are also overlapped. Thus, studying the biological effects of microsomal or soluble EH alterations needs to include the effects on both the metabolism of reactive metabolites, as well as epoxides of fatty acids. This review focuses on the multifaceted role of EH in the metabolism of xenobiotic and endogenous epoxides and the impact of EH modulations.
... Oleamide induces vasorelaxation in rat mesenteric arteries in a manner similar to arachidonoylethanolamide (Hoi and Hiley, 2006) – another example of the cannabinoid-like effects of oleamide. The trans isomer of oleamide, elaidamide, is a potent inhibitor of the xenobiotic-metabolizing enzyme microsomal epoxide hydrolase (mEH) (Morisseau et al., 2001). The inhibition of this enzyme by elaidamide resulted in enhanced toxicity of the epoxide-containing xenobiotic cis-stilbene oxide (Morisseau et al., 2001). ...
... The trans isomer of oleamide, elaidamide, is a potent inhibitor of the xenobiotic-metabolizing enzyme microsomal epoxide hydrolase (mEH) (Morisseau et al., 2001). The inhibition of this enzyme by elaidamide resulted in enhanced toxicity of the epoxide-containing xenobiotic cis-stilbene oxide (Morisseau et al., 2001). Elaidamide possesses structural similarities to the known mEH inhibitor valpromide. ...
The golden alga Prymnesium parvum has been implicated in fish and aquatic animal kills globally for over a century. In addition to widespread ecological impacts through the loss of entire fish populations within lakes, an economic burden is also felt by state and local agencies due to year class losses of fish raised for stocking lakes as well as loss of fishing and recreational use of the affected water. Multiple compounds have been implicated in P. parvum toxicity, but the unequivocal identification and characterization of all P. parvum toxins remained to be accomplished. To unambiguously characterize these toxins, we analyzed laboratory-cultured cells exposed to limited nitrogen and phosphorus concentrations, uni-algal wild cells collected from an ichthytoxic bloom event at Lake Wichita, TX, and the water from both cultured and field-collected algae. A bioassay-guided fractionation process was employed to chemically isolate P. parvum toxins using both mammalian cells and larval fish. The results of these assays revealed that there was a distinct similarity in the toxic compounds characterized as seven primary fatty acid amides (myristamide, palmitamide, linoleamide, oleamide, elaidamide, stearamide, and erucamide) and one hydroxamic acid (linoleyl hydroxamic acid). These compounds display cytotoxic and ichthytoxic activity and have not yet been reported in P. parvum toxicity or in the toxicity of harmful algal species.
... Expression of human mEH in a recombinant baculovirus system (Morisseau et al., 2001) and the purification procedure of the solubilized form of mEH (Akatsuka et al., 2007) have been described. Briefly, the infected cells were solubilized with Triton X-100 and subjected to Q-Sepharose column chromatography. ...
... Concentrated culture media were dissolved 10 folds in Tris/HCl buffer (0.1 M pH 9.0) containing 0.1 mg/ml BSA and the activities were measured with [ 3 H]-cis-stilbene oxide after incubation for 15 min at 30°C (Morisseau et al., 2001). Purified human mEH (S-mEH) diluted 100-folds was used as positive control, and protein content was measured with BCA assay (Pierce, Rockford, IL) using BSA as standard. ...
Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases.
... In another study, a highly enatio-selective EH from Nocardia sp. was purified to homogeneity by using a combination of four chromatographic steps that included hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, anion exchange chromatography on SOURCE 30Q, followed by a second hydrophobic interaction chromatography on Phenyl Sepharose HP and gel-filtration on Superdex 75 HR to remove the unwanted proteins 82 . Previously, an EH was also successfully purified from Rhodococcus erythropolis strain DCL14 was originally isolated from a fresh-water sediment sample 83,84 . ...
In the last two decades the exploitation of enzyme and microbes by synthetic as well as pharmaceutical industry has increased substantially. Epoxide hydrolase (EH) is an important enzyme widely used in kinetic resolution and synthesis of vicinal diol. This a highly attractive biocatalyst used in the formation of a single enantiomeric diol from a racemic oxirane. The microbial epoxide hydrolase hydrolyses substrates of various structural types. EHs are cofactor-independent enzymes that are easy to use for organic synthesis. Moreover, these enzymes are ubiquitous and not restricted to the mammalian world only. These are found in bacteria, yeast, fungi, plants and insects. There is a wide range of applications of EHs in pharmaceuticals as well as in clinical industry. EHs may enable the preparation of enantiopure epoxides in a very simple way starting from cheap and easily available racemic epoxides. This review covers the structure, mechanism of action and catalytic potential of EHs in pharmacological and synthetic industry.
... Although this compound possessed proper efficacy in vitro, fast metabolism by amidase limited its use in vivo. Therefore, the necessity for new potent and stable sEHIs continued (Fig. 3) [50]. ...
Soluble epoxide hydrolase is a class of α/β-fold hydrolase enzymes that exist in numerous organs and tissues, including the liver, kidney, brain, and vasculature. This homodimer enzyme is responsible for degrading epoxyeicosatrienoic acids to the less active vicinal diols, dihydroxyeicosatrienoic acids through adding a molecule of water to an epoxide in the cytochrome P450 pathway. Soluble epoxide hydrolase was firstly assayed and characterized by Hammock and colleagues about 40 years ago. Upholding high epoxyeicosatrienoic acid blood levels by inhibiting soluble epoxide hydrolase has been proposed as a hopeful strategy to treat renal and cardiovascular diseases, inflammation, and pain. Therefore, developing novel soluble epoxide hydrolase inhibitors has been an attractive research topic for many years. Regarding this issue, some carbamates, heterocycles, amides, and ureas have been proposed; however, rapid metabolism, low solubility, high melting point, and weak pharmacokinetic characteristics are challenges posed to the researchers. In this review, we have focused on the role of the soluble epoxide hydrolase in the metabolic pathway of arachidonic acid, and categorized most representative soluble epoxide hydrolase inhibitors into two main classes of synthetic and natural compounds. The structures have been evaluated and an exemplary structure-activity relationship has been provided for further development of potent inhibitors at the end. According to our findings, urea-based inhibitors were preferred to the amide-based scaffolds due to the better fitting into the active site. An aromatic linker is a suitable bridge to connect primary and secondary pharmacophores compared with aliphatic linkers.
... Structural formulas for these inhibitors can be found in previous reports [24]. More potent amide-based EPHX1 inhibitors were also engineered, and inhibit EPHX1 activity by associating to the catalytic domain with low nanomolar dissociation [53,54]. EPHX1 inhibition was reported to be beneficial in the case of PAH exposure, and 1,1,1-trichloropropene-2,3-oxide reduced the carcinogenicity of DMBA [55]. ...
Epoxide hydrolases (EHs) are key enzymes involved in the detoxification of xenobiotics and biotransformation of endogenous epoxides. They catalyze the hydrolysis of highly reactive epoxides to less reactive diols. EHs thereby orchestrate crucial signaling pathways for cell homeostasis. The EH family comprises 5 proteins and 2 candidate members, for which the corresponding genes are not yet identified. Although the first EHs were identified more than 30 years ago, the full spectrum of their substrates and associated biological functions remain partly unknown. The two best-known EHs are EPHX1 and EPHX2. Their wide expression pattern and multiple functions led to the development of specific inhibitors. This review summarizes the most important points regarding the current knowledge on this protein family and highlights the particularities of each EH. These different enzymes can be distinguished by their expression pattern, spectrum of associated substrates, sub-cellular localization, and enzymatic characteristics. We also reevaluated the pathogenicity of previously reported variants in genes that encode EHs and are involved in multiple disorders, in light of large datasets that were made available due to the broad development of next generation sequencing. Although association studies underline the pleiotropic and crucial role of EHs, no data on high-effect variants are confirmed to date.
... COX enzyme proved to be difficult to purify and was not sequenced until 1988. 35 But in 1991 the COX-2 enzyme was cloned and its existence, therefore, confirmed. Before the confirmed existence of COX-2, the Dupont Company had developed a Compound, DuP-697, which was potent in many anti-inflammatory assays but did not have the ulcerogenic effects of NSAIDs. ...
NSAIDs play an important role in the treatment of inflammation, however due to
their GI side effect on long term use; their use has been restricted in the treatment of
arthritis. Discovery of the COX isoforms and understanding their individual role in
inflammation led to the concept of selective COX-2 inhibitors. Celecoxib is the most
potent selective COX-2 inhibitor, however due to its cardiovascular complications its use
has been restricted. sEH is an enzyme responsible for conversion of epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). EETs are an endogenous anti-hypertensive eicosanoids. Thus inhibiting the sEH increase the level of EETs which in turn produce vasodilation and thus help to overcome the side effect of selective COX-2 inhibitor. In an attempt to design new drugs for the treatment of inflammation with a better safety profile and efficacy, a series of novel, potent dual COX-2 and sEH inhibitors were designed and synthesized. Molecular Docking studies of the designed dual inhibitors are done using 1CX2 for COX-2 and 1ZD5 for sEH as a targeted protein. All the designed dual inhibitors have shown promising results in docking studies. The synthesized compounds are submitted for their biological evaluation; the assay will be performed using recombinant enzyme kit
... The ABHD is commonly associated with esterase and lipase activities (19)(20)(21)(22). Interestingly, previous structure-function studies in EPHX1 inhibition demonstrated that a number of long-chain FA derivatives or valproic acid derivatives are potent, partially competitive inhibitors of EPHX1-mediated hydrolysis of cis -stilbene oxide (cSO) or (S)-(+) styrene oxide, well-known EPHX1 substrates (23)(24)(25). The results suggest that these compounds can bind to EPHX1 and may modify and/or interrupt the catalytic sites for the substrates. ...
Microsomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a highly abundant α/β hydrolase (ABHD) enzyme which is known for its catalytical epoxide hydrolase activity. A wide range of EPHX1 functions has been demonstrated including xenobiotic metabolism; however, characterization of its endogenous substrates is limited. In this study, we present evidence that EPHX1 metabolizes the abundant endocannabinoid, 2-arachidonoylglycerol (2-AG) to free arachidonic acid (AA) and glycerol. The EPHX1 metabolism of 2-AG was demonstrated using commercially available EPHX1 microsomes as well as PC-3 cells overexpressing EPHX1. Conversely, EPHX1 siRNA markedly reduced the EPHX1 expression and 2-AG metabolism in HepG2 cells and LNCaP cells. A selective EPHX1 inhibitor, 10-hydroxystearamide (10-HSA), inhibited 2-AG metabolism and hydrolysis of a well-known EPHX1 substrate, cis-stilbene oxide (cSO). Among the inhibitors studied, a serine hydrolase inhibitor, methoxy-arachidonyl fluorophosphate (MAFP) was the most potent inhibitor of 2-AG metabolism by EPHX1 microsomes. These results demonstrate that 2-AG is an endogenous substrate for EPHX1, a potential role of EPHX1 in the endocannabinoid signaling and a new AA biosynthetic pathway.
... Me (Morisseau et al., 2001;Kim et al., 2005;Campbell et al., 1996). Epoxyeicosatrienoic acids (EETs) are formed in mammalian tissues by epoxygenases of the cytochrome P450 (CYP) superfamily that use arachidonic acid as their substrates. ...
A series of novel sEH inhibitors containing a N-substituted piperidine ring [N-urea (8a–i) (9a–l) and amide derivatives (6a–l) (7a–l)] with pyrazole (a five-membered polar heterocycle) as a pharmacophore lead for sEH inhibition have been designed, synthesized and evaluated as novel analogues to reduce blood pressure elevation and inflammatory roles by acting as sEH inhibitors. The synthesized compound shows varying degree of selectivity towards the sEH enzymes. Particularly compounds 9g and 8h emerged as the most potent sEH inhibitor displaying IC50 values of 0.224 ± 0.014 and 0.220 ± 0.03 μM for in vitro sEH inhibition. Molecular docking studies of the designed sEH dual inhibitors are performed using 1ZD5 for sEH as the targeted proteins and which revealed H-bond interactions similar to AUDA. Structure–activity relationships provided useful insights in these classes of compounds and paved the way to design novel analogues with increased potency.
Graphical abstract
Schematic representation of proposed sEH Inhibitors.
... However, all of these inhibitors are substrates and provide only temporary inhibition of mEH activity before being turned over. Recently, Morisseau et al. have studied the inhibitory potential of fatty amides, and they found elaidamide to be the most potent inhibitor with Ki value of 70 nM (Morisseau et al., 2001). However, in an in vivo context, this class of inhibitors was rapidly metabolized to an inactive form. ...
Microsomal epoxide hydrolase (mEH) is a liver enzyme involved in hepatic and extrahepatic metabolism of xenobiotics, namely, the hydrolysis of epoxides and arene oxides to the corresponding diols. In some cases, the action of mEH activates xenobiotics, such as 7,12-dimethylbenz[a]anthracene, potentiating their ability to induce mammary, ovarian, skin, and other types of cancer according to Rajapaksa et al. (Toxicol. Sci 96:327–334, 2006). Similarly, mEH polymorphisms have been linked to several types of cancer as stated by Benhamou et al. (Cancer Res. 58:5291–5293, 1998), and also to emphysema according to Smith and Harrison (Lancet 350:630–633, 1997). mEH inhibitors would provide insight into the multiple roles of this enzyme and potentially have clinical relevance for preventing disease in high-risk individuals. In this article, we describe the design and synthesis of acylthiourea analogs as inhibitors of mEH.
... Glycidol-ester and epoxy-ester analogs of methyl 10,11-epoxy-11-methyldodecanoate (MEMD) are potent inhibitors of the JHEH, but they may not be specific to insects. 97) Recently, amide-, urea-and carbamate-type analogs have been reported as potential EH inhibitors in mammals, 98,99) and some potent analogs inhibited JHEH activity in vitro. 100) Interestingly, most of the inhibitors of the mammalian EHs are not effective on insect JHEH, 100) but converse studies of inhibitors for JHEH that do not act on the mammalian enzyme have not been reported. ...
Juvenile hormone (JH) is a sesquiterpenoid hormone that regulates growth and development in insects. Since JH is a hormone specific to insects and other arthropods, compounds disrupting JH action in insects are expected to be ideal insecticides with low toxicity to non-target organisms. Many natural or synthetic analogs with JH-like or anti-JH activity have been identified, and some potent JH mimics have been used as insecticides. Recent studies on the enzymes in JH biosynthetic and metabolic pathways should be helpful for the discovery of more potent analogs and for the establishment of new means of pest management using recombinant DNA technology. 0 Pesticide Science Society of Japan.
... Inhibitor assays using fluorescent substrates-The half maximal inhibitory concentration (IC 50 ) of JH-like compounds (ureas, secondary amides, and primary amides) and non-JH-like primary amide compounds against Hv-mEH1, MsJHEH, and hmEH were determined using the fluorescent assays described above. The methods used to synthesize and determine the physical characteristics of these compounds are previously described (Morisseau et al., 1999;Morisseau et al., 2001;Morisseau et al., 2002;Severson et al., 2002;Morisseau et al., 2011). Each potential inhibitor was preincubated with the enzyme for 5 min at 30°C prior to the addition of substrate. ...
Epoxide hydrolases (EHs) are α/β-hydrolase fold superfamily enzymes that convert epoxides to 1,2-trans diols. In insects EHs play critical roles in the metabolism of toxic compounds and allelochemicals found in the diet and for the regulation of endogenous juvenile hormones (JHs). In this study we obtained a full-length cDNA, hvmeh1, from the generalist feeder Heliothis virescens that encoded a highly active EH, Hv-mEH1. Of the 10 different EH substrates that were tested, Hv-mEH1 showed the highest specific activity (1,180 nmol min(-1) mg(-1)) for a 1,2-disubstituted epoxide-containing fluorescent substrate. This specific activity was more than 25- and 3,900-fold higher than that for the general EH substrates cis-stilbene oxide and trans-stilbene oxide, respectively. Although phylogenetic analysis placed Hv-mEH1 in a clade with some lepidopteran JH metabolizing EHs (JHEHs), JH III was a relatively poor substrate for Hv-mEH1. Hv-mEH1 showed a unique substrate selectivity profile for the substrates tested in comparison to those of MsJHEH, a well-characterized JHEH from M. sexta, and hmEH, a human microsomal EH. Hv-mEH1 also showed unique enzyme inhibition profiles to JH-like urea, JH-like secondary amide, JH-like primary amide, and non-JH-like primary amide compounds in comparison to MsJHEH and hmEH. Although Hv-mEH1 is capable of metabolizing JH III, our findings suggest that this enzymatic activity does not play a significant role in the metabolism of JH in the caterpillar. The ability of Hv-mEH1 to rapidly hydrolyze 1,2-disubstituted epoxides suggests that it may play roles in the metabolism of fatty acid epoxides such as those that are commonly found in the diet of Heliothis.
... The sample was then heated in 49°C water bath for 5 min before angiogenesis ( 25 ) and controls water balance ( 26 ). In addition, both mammalian phospholipase A 2 (PLA 2 ) and epoxide hydrolase (EH) are inhibited by series of PFAMs, with arachidonamide and ␥ -linolenamide being the most potent for the inhibition of PLA 2 ( 27 ) and elaidamide being the most potent for the inhibition of EH ( 28 ). The physiological signifi cance of the reported functions of oleamide and the other PFAMs is not completely clear as relatively high concentrations are sometimes used to elicit the indicated responses. ...
Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.
... -Inhibitors of EHs: CDU (0.01 M, 0.1 M and 1 M), as a sEH inhibitor (Davis et al., 2006), elaidamide (10 M), as a mEH inhibitor (Morisseau et al., 2001). Zn 2+ (10 mM), was also used as an inhibitor of both EHs (Draper and Hammock, 1999), though it is also known that this metal may behave as an antioxidant; -Antioxidant: NAC (500 M), as an antioxidant agent (Kligerman and Tennant, 2007); -Mitochondrial protective agents: ALC (5 mM), a potent restorer of mitochondria energy production (Boerrigter et al., 1993) and ␣-LA (100 M), a restorer of mitochondrial redox status (Han et al., 1997). ...
The biotransformation and oxidative stress may contribute to 1,2:3,4-diepoxybutane (DEB)-induced toxicity to human lymphocytes of Fanconi Anemia (FA) patients. Thus, the identification of putative inhibitors of bioactivation, as well as the determination of the protective role of oxidant defenses, on DEB-induced toxicity, can help to understand what is failing in FA cells. In the present work we studied the contribution of several biochemical pathways for DEB-induced acute toxicity in human lymphocyte suspensions, by using inhibitors of epoxide hydrolases, inhibitors of protective enzymes as glutathione S-transferase and catalase, the depletion of glutathione (GSH), and the inhibition of protein synthesis; and a variety of putative protective compounds, including antioxidants, and mitochondrial protective agents. The present study reports two novel findings: (i) it was clearly evidenced, for the first time, that the acute exposure of freshly isolated human lymphocytes to DEB results in severe GSH depletion and loss of ATP, followed by cell death; (ii) acetyl-l-carnitine elicits a significant protective effect on DEB induced toxicity, which was potentiated by α-lipoic acid. Collectively, these findings contribute to increase our knowledge of DEB-induce toxicity and will be very useful when applied in studies with lymphocytes from FA patients, in order to find out a protective agent against spontaneous and DEB-induced chromosome instability.
... Chemicals-3-Morpholinosydnonimine hydrochloride (SIN-1), 14,15-EET, 14,15-d8-EET, and recombinant human sEH were from Cayman Chemicals (Ann Arbor, MI) and NADPH from AppliChem (Darmstadt, Germany). The sEH inhibitor 1-adamantyl-3-cyclohexylurea (ACU) and the microsomal epoxide hydrolase inhibitor elaidamide were synthesized as described (12,13). The anti-human sEH antibody used was purified by Eurogenetec (Seraing, Belgium) from rabbits immunized with two sEH peptides (H 2 N-KGYGESSAPPEIEEYC-CONH 2 and H 2 N-CGHWTQMDKPTEV-CONH 2 ), and the polyclonal anti-mouse sEH antibody (dilution of 1:2000) against a recombinant murine sEH produced in a baculovirus expression system and then purified to apparent homogeneity by affinity chromatography. ...
Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr(383) and Tyr(466)) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO(-)) or the ONOO(-) generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO(-) and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr(383) and Tyr(466). Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.
... 1,1,1,-Trichloropropene-2,3-oxide was found to be an inhibitor of rodent and human mEH (Papadopoulos et al. 1985) and cyclopropyl oxiranes are competitive reversible inhibitors for mEH (Prestwich et al. 1985) (Fig. 4c). Also the anticonvulsant valpromide was identiWed as a mEH inhibitor and recently other substituted fatty acid amides (elaidamide) were developed as metabolically stable mEH inhibitors of much higher aYnity (Morisseau et al. 2001(Morisseau et al. , 2008. ...
Epoxide hydrolases catalyse the hydrolysis of electrophilic--and therefore potentially genotoxic--epoxides to the corresponding less reactive vicinal diols, which explains the classification of epoxide hydrolases as typical detoxifying enzymes. The best example is mammalian microsomal epoxide hydrolase (mEH)-an enzyme prone to detoxification-due to a high expression level in the liver, a broad substrate selectivity, as well as inducibility by foreign compounds. The mEH is capable of inactivating a large number of structurally different, highly reactive epoxides and hence is an important part of the enzymatic defence of our organism against adverse effects of foreign compounds. Furthermore, evidence is accumulating that mammalian epoxide hydrolases play physiological roles other than detoxification, particularly through involvement in signalling processes. This certainly holds true for soluble epoxide hydrolase (sEH) whose main function seems to be the turnover of lipid derived epoxides, which are signalling lipids with diverse functions in regulatory processes, such as control of blood pressure, inflammatory processes, cell proliferation and nociception. In recent years, the sEH has attracted attention as a promising target for pharmacological inhibition to treat hypertension and possibly other diseases. Recently, new hitherto uncharacterised epoxide hydrolases could be identified in mammals by genome analysis. The expression pattern and substrate selectivity of these new epoxide hydrolases suggests their participation in signalling processes rather than a role in detoxification. Taken together, epoxide hydrolases (1) play a central role in the detoxification of genotoxic epoxides and (2) have an important function in the regulation of physiological processes by the control of signalling molecules with an epoxide structure.
... DHETs were initially thought to be inactivation products of EETs, but several recent studies indicate that, like EETs, they produce vasodilation and activate smooth muscle BK Ca channels [35][36][37][38]. There are two major epoxide hydrolase isozymes in mammalian tissues; soluble epoxide hydrolase (sEH) contained primarily in the cytosol [33,34], and microsomal epoxide hydrolase (mEH) bound to intracellular membranes [39]. sEH is the main isozyme that acts on the EETs. ...
Epoxyeicosatrienoic acids (EETs), which are synthesized from arachidonic acid by cytochrome P450 epoxygenases, function primarily as autocrine and paracrine effectors in the cardiovascular system and kidney. They modulate ion transport and gene expression, producing vasorelaxation as well as anti-inflammatory and pro-fibrinolytic effects. EETs are incorporated into the sn-2 position of phospholipids and are rapidly mobilized when a cell is treated with a Ca(2+) ionophore, suggesting that they may play a role in phospholipid-mediated signal transduction processes. Soluble epoxide hydrolase (sEH) converts EETs to dihydroxyeicosatrienoic acids (DHETs), and inhibition of sEH is a potential approach for enhancing the biological activity of EETs. EETs also undergo chain-elongation and beta-oxidation, and the accumulation of partial beta-oxidation products increases when sEH is inhibited. Some functional effects of EETs occur through activation of either the guanine nucleotide binding protein Galphas or the Src signal transduction pathways, suggesting that EETs act by binding to membrane receptors. However, other evidence indicates that the modulation of gene expression occurs through an intracellular action of EETs. Because of the diversity of biochemical and functional responses produced by EETs, it is doubtful that a single mechanism or signal transduction pathway can account for all of their actions.
Background:
The prognosis of hepatocellular carcinoma (HCC) patients remains poor despite advances in treatment modalities and diagnosis. It is important to identify useful markers for the early detection of HCC in patients. Preneoplastic antigen (PNA), originally reported in a rat carcinogenesis model, is increased in the tissues and serum of HCC patients.
Aim:
To determine the diagnostic value of PNA for discriminating HCC and to characterize PNA-positive HCC.
Methods:
Patients with hepatitis C virus (HCV)-related hepatic disorders were prospectively enrolled in this study, which included patients with hepatitis, with cirrhosis, and with HCC. A novel enzyme-linked immunosorbent assay was developed to measure serum PNA concentrations in patients.
Results:
Serum PNA concentrations were measured in 89 controls and 141 patients with HCV infections (50 hepatitis, 44 cirrhosis, and 47 HCC). Compared with control and non-HCC patients, PNA was increased in HCC. On receiver operating characteristic curve analysis, the sensitivity of PNA was similar to the HCC markers des-γ-carboxy-prothrombin (DCP) and α-fetoprotein (AFP), but the specificity of PNA was lower. There was no correlation between PNA and AFP and a significant but weak correlation between PNA and DCP in HCC patients. Importantly, the correlations with biochemical markers were completely different for PNA, AFP, and DCP; glutamyl transpeptidase was highly correlated with PNA, but not with AFP or DCP, and was significantly higher in PNA-high patients than in PNA-low patients with HCV-related HCC.
Conclusion:
PNA may have the potential to diagnose a novel type of HCC in which glutamyl transpeptidase is positively expressed but AFP or DCP is weakly or negatively expressed.
Epoxide hydrolases (EHs) metabolize highly reactive epoxides with mutagenic and carcinogenic potential to the less reactive corresponding diols and are therefore traditionally viewed as detoxicating enzymes. In few instances, however, the diols themselves can be precursors of further reactive metabolites, and EH may thereby contribute to metabolic toxification. The most important xenobiotic-metabolizing EH is the microsomal EH (mEH). Furthermore, evidence is emerging that mammalian EHs fulfill roles other than detoxication. Meanwhile, the role of soluble EH (sEH), whose physiological substrates are epoxyeicosanoids (epoxyeicosatrienoic acids, EETs), signaling molecules involved in a broad variety of regulatory pathways, is well documented. sEH is therefore considered a new drug target as respective inhibitors promise therapeutic potential in the treatment of hypertension, pain, and possibly other diseases. Two new human EHs, EH3 and EH4, have been recently identified. Their expression pattern and substrate specificity strongly suggest a role in physiological processes, similar to sEH, rather than in metabolizing xenobiotic compounds.
Significance
This study furthers our understanding of epoxyeicosatrienoic acid metabolism by cyclooxygenase (COX) enzymes as a physiologically relevant metabolic pathway, producing signaling molecules that are angiogenic. It explains, in part, why inhibiting the soluble epoxide hydrolase (sEH) in some systems is angiogenic whereas combining sEH inhibition with COX inhibition is dramatically antiangiogenic, which in turn may suppress tumor growth.
The present study presents comprehensive chemical analyses of cuticular wax mixtures of the bamboo Phyllostachys aurea. The epicuticular and intracuticular waxes were sampled selectively from the adaxial side of leaves on young and old plants and investigated by gas chromatography-mass spectrometry and flame ionization detection. The epi- and intracuticular layers on young and old leaves had wax loads ranging from 1.7 μg/cm(2) to 1.9 μg/cm(2). Typical very-long-chain aliphatic wax constituents were found with characteristic chain length patterns, including alkyl esters (primarily C48), alkanes (primarily C29), fatty acids (primarily C28 and C16), primary alcohols (primarily C28) and aldehydes (primarily C30). Alicyclic wax components were identified as tocopherols and triterpenoids, including substantial amounts of triterpenoid esters. Alkyl esters, alkanes, fatty acids and aldehydes were found in greater amounts in the epicuticular layer, while primary alcohols and most terpenoids accumulated more in the intracuticular wax. Alkyl esters occurred as mixtures of metamers, combining C20 alcohol with various acids into shorter ester homologs (C36C40), and a wide range of alcohols with C22 and C24 acids into longer esters (C42C52). Primary amides were identified, with a characteristic chain length profile peaking at C30. The amides were present exclusively in the epicuticular layer and thus at or near the surface, where they may affect plant-herbivore or plant-pathogen interactions.
The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. Based on these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif, and have the potential for clinical application.
Epoxide hydrolases (EHs) metabolize highly reactive epoxides with mutagenic and carcinogenic potential to the less reactive corresponing diols and are therefore traditionally viewed as detoxicating enzymes. In few instances, however, the diols themselves can be precursors of further reactive metabolites, and EH may thereby contribute to metabolic toxification. The most important xenobiotic-metabolizing EH is the microsomal EH (mEH). Furthermore, Evidence is emerging that mammalian EHs fulfill roles other than detoxication. Meanwhile, the role of soluble EH (sEH), whose physiological substrates are epoxyeicosanoids (epoxyeicosatrienoic acids, EETs), signaling molecules involved in a broad variety of regulatory pathways is well documented. sEH is therefore considered a new drug target as respective inhibitors promise therapeutic potential in the treatment of hypertension and possibly other diseases. Two new human EHs have been recently identified by our group. Their expression pattern and substrate specificity strongly suggest a role in physiological processes similar to one of the sEH rather than in metabolizing xenobiotic compounds.
Fatty acid amides have emerged as an intriguing family of diverse, mammalian neuroactive lipids. Herein, we review the current state of our knowledge about the individual classes of fatty acid amides: which ones have been identified and characterized from mammals, their receptors, their functions, and the pathways for their biosynthesis and degradation. Much remains to be elucidated regarding this family of molecules and we hope that our review stimulates additional research in the fatty acid amide field. We also show that there are extensive metabolic connections between the different classess of fatty acid amides. Such metabolic connections hint at novel modes of regulation as one fatty acid amide is converted to another and suggests that the enzymes involved in fatty acid amide metabolism are not simply enzymes of biosynthesis or degradation, but also serve important regulatory functions.
Epoxides are organic three-membered cyclic oxygen compounds that derive from oxidative metabolism of endogenous metabolites (e.g., intermediate in cholesterol biosynthesis), as well as xenobiotic compounds (e.g., metabolite of benzpyrene), via chemical and enzymatic oxidation processes. The resultant unstable, chemically reactive epoxides are known to be mutagenic and carcinogenic initiators. Therefore, it is of vital importance to regulate levels of these reactive species. Epoxide hydrolase, also known as Arene-oxyde hydratase, catalyzes the hydrolysis of arene and aliphatic epoxides to less reactive and more water-soluble dihydrodiols by the trans addition of water. The following general equation characterizes these enzymes: H 2O + epoxide = glycol. There are two epoxide hydrolase subtypes: The microsomal, membrane bound (mEH, EPHX1) and the soluble, cytosolic form (sEH, EPHX2).
Antiöstrogene wurden in den letzten Jahren sehr effektiv in der Therapie des Mammakarzinoms eingesetzt. Im Verlauf der Therapie kann es jedoch zur Ausbildung von Tamoxifenresistenzen kommen, die den Ausgang negativ beeinflussen. Die funktionellen Mechanismen, die zur Entstehung dieser Resistenz führen, sind bisher noch weitgehend unbekannt.
Grundlage dieser Arbeit ist eine Studie, in der gezeigt wurde, dass eine hohe Expression der mikrosomalen Epoxidhydrolase (mEH) zu einem schlechteren Verlauf der Tamoxifentherapie bei Patientinnen mit Mammakarzinom führt (Fritz et al. 2001). Dies wiederum lässt die Vermutung zu, dass eine erhöhte mEH-Expression zu einer Tamoxifenresistenz führen könnte. Eine funktionelle Analyse der möglichen Mechanismen hinter dieser Beobachtung könnte zu einem besseren Verständnis der Resistenzausbildung und somit zu möglichen neuen Therapieansätzen führen.
Im ersten Teil der Arbeit wurden hormonsensitive MCF-7 Mammakarzinom Zelllinien eingesetzt, um den möglichen Einfluss der mEH auf die antiestrogen binding site (AEBS) zu untersuchen. Die Ergebnisse zeigten, dass nach Behandlung mit Tamoxifen und dem AEBS-Inhibitor DPPE keine Änderung des Zellwachstums beobachtet werden konnte. Somit hat die mEH als Untereinheit der AEBS keinen Einfluss auf die Tamoxifenwirkung.
Auf Grund der kompetitiven Wirkung von Östrogenen und Antiöstrogenen, könnte mEH die Tamoxifenbehandlung auf indirektem Weg über den Östradiol-(βE2-)signalweg beeinflussen. Um dies zu untersuchen wurde ein ERE-(estrogen response element)-Reportergenkonstrukt verwendet und die βE2-Antwort nach Behandlung mit Tamoxifen oder βE2 und Transfektion von mEH oder mEH_6C (verkürzte mEH, die durch ein früheres Stop-Codon kein aktives Zentrum beinhaltet) untersucht. Es zeigte sich eine Sensitivierung gegenüber βE2 nach Überexpression von mEH_6C. Des Weiteren konnte nach Inhibition der mEH mittels siRNA eine Hemmung der βE2-Antwort beobachtet werde. Analysen der mRNA von pS2 (βE2-Zielgen) und dem Östrogenrezeptor α (ERα) zeigten eine Inhibition in Abwesenheit der mEH.
In einem weiteren, hormonell regulierten System, dem Corpusendometrium der Frau, sollte eine mögliche hormonelle Regulation der mEH innerhalb des Menstruationszyklus untersucht werden. Es zeigte sich nach Analyse der Paraffinschnitte eine signifikante Erhöhung der mEH-Expression im Verlauf des Zyklus. Während der Schwangerschaft erreichte diese ihren Höhepunkt.
Da die mEH-Expression Ähnlichkeiten zur Progesteronexpression im Verlauf des Zyklus aufweist, wurde im Zellkultursystem eine mögliche Regulation der mEH durch Progesteron (Medroxy Progesteronacetat, MPA) untersucht. Anhand von zwei Endometrium-Zelllinien, der ERα-positiven (ERα+) ECC1PRAB-72 (PRA+, PRB+) und der ERα-negativen (ERα-) IKPRAB-36 (PRA+, PRB+) konnte gezeigt werden, dass MPA die mEH-Expression in Abhängigkeit des ERα hoch reguliert.
Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass mEH einen regulatorischen Einfluss auf die βE2-Antwort ausübt und gleichzeitig die mEH-Expression durch Progesteron reguliert wird. Somit scheint die mEH eine bis dahin noch unbekannte Funktion im hormonellen System auszuüben, die eine wichtige Rolle spielen könnte und möglicherweise einen neuen Ansatz zur Überwindung der Antiöstrogenresistenz des Mammakarzinoms liefern könnte.
Protein engineering aimed at enhancing enzyme stability is increasingly supported by computational methods for calculation of mutant folding energies and for the design of disulfide bonds. To examine the accuracy of mutant structure predictions underlying these computational methods, crystal structures of thermostable limonene epoxide hydrolase variants obtained by computational library design were determined. Four different predicted effects indeed contributed to the obtained stabilization: (i) enhanced interactions between a flexible loop close to the N-terminus and the rest of the protein; (ii) improved interactions at the dimer interface; (iii) removal of unsatisfied hydrogen bonding groups; and (iv) introduction of additional positively charged groups at the surface. The structures of an eightfold and an elevenfold mutant showed that most mutations introduced the intended stabilizing interactions, and side-chain conformations were correctly predicted for 72-88% of the point mutations. However, mutations that introduced a disulfide bond in a flexible region had a larger influence on the backbone conformation than predicted. The enzyme active sites were unaltered, in agreement with the observed preservation of catalytic activities. The structures also revealed how a c-Myc tag, which was introduced for facile detection and purification, can reduce access to the active site and thereby lower the catalytic activity. Finally, sequence analysis showed that comprehensive mutant energy calculations discovered stabilizing mutations that are not proposed by the consensus or B-FIT methods. This article is protected by copyright. All rights reserved.
© 2015 Wiley Periodicals, Inc.
A series of novel dual inhibitor of substituted 9-oxo-1,2-dihydropyrrolo[2,1-b]quinazolin-3(9H)-ylidene)methyl)piperidine-1-carboxamide Derivatives as a pharmacophore lead for Potent antiinflammatory and sEH inhibition have been designed, synthesized and evaluated as novel analogues to act as selective COX-2 Inhibitors over COX-1 which prevent blood pressure elevation by acting as sEH inhibitors in addition. The synthesized compounds 10e and 10g exhibit varying degrees COX-2 selectivity and inhibition of sEH enzyme displaying IC 50 values of 0.124±0.011µM and 0.110±0.01µM for in-vitro sEH inhibition respectively.
Unexplained deaths of pond-grown catfish have occurred for many years. At least some of these mortalities could be from cyanobacteria toxins ingested during feeding on floating diets or passively assimilated through gills during breathing. Recently we were able to document algal production and subsequent ingestion of these toxicants by catfish during a mortality event. The causative organism, Microcystis aeruginosa, was the dominant species within the phytoplankton community during the cooler autumn-winter season. Pond conditions included a drop in water temperature by c. 5 °C during the 10 days preceding the fish mortalities. Microcystin-LR, a hepatotoxin produced by Microcystis, was detected in water samples and in catfish liver tissue. Fish exposed to pond water containing this toxic bloom were killed within 24 h. Necropsy of fish revealed congested liver and spleen tissues. The combination of clinical signs, detection of microcystin LR in water and in liver, and death of fish exposed to pond water supports the diagnosis of microcystin toxicosis. More research is needed to identify specific environmental conditions initiating toxin production to model and predict occurrence of these toxic algal blooms.
A sensitive, competitive indirect enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclocarban (TCC) was developed. The haptens were synthesized by derivatizing the para position of a phenyl moiety of TCC. The rabbit antisera were screened and the combination of antiserum 1648 and a heterologous competitive hapten containing a piperidine was further characterized. The IC(50) and detection range for TCC in buffer were 0.70 and 0.13-3.60 ng/mL, respectively. The assay was selective for TCC, providing only low cross-reactivity to TCC-related compounds and its major metabolites except for the closely related antimicrobial 3-trifluoromethyl-4,4'-dichlorocarbanilide. A liquid-liquid extraction for sample preparation of human body fluids resulted in an assay that measured low part per billion levels of TCC in small volumes of the samples. The limits of quantification of TCC were 5 ng/mL in blood/serum and 10 ng/mL in urine, respectively. TCC in human urine was largely the N- or N'-glucuronide. TCC concentrations of biosolids measured by the ELISA were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid, inexpensive, and convenient tool to aid researchers monitoring human/environmental exposure to TCC to better understand the health effects.
3,3-Disubstituted piperidine-derived trisubstituted urea entA-2b was discovered as a highly potent and selective soluble epoxide hydrolase (sEH) inhibitor. Despite the good compound oral exposure, excellent sEH inhibition in whole blood, and remarkable selectivity, compound entA-2b failed to lower blood pressure acutely in spontaneously hypertensive rats (SHRs). This observation further challenges the premise that sEH inhibition can provide a viable approach to the treatment of hypertensive patients.
All-cis-14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a labile, vasodilatory eicosanoid generated from arachidonic acid by cytochrome P450 epoxygenases. A series of robust, partially saturated analogues containing epoxide bioisosteres were synthesized and evaluated for relaxation of precontracted bovine coronary artery rings and for in vitro inhibition of soluble epoxide hydrolase (sEH). Depending upon the bioisostere and its position along the carbon chain, varying levels of vascular relaxation and/or sEH inhibition were observed. For example, oxamide 16 and N-iPr-amide 20 were comparable (ED(50) 1.7 microM) to 14,15-EET as vasorelaxants but were approximately 10-35 times less potent as sEH inhibitors (IC(50) 59 and 19 microM, respectively); unsubstituted urea 12 showed useful activity in both assays (ED(50) 3.5 microM, IC(50) 16 nM). These data reveal differential structural parameters for the two pharmacophores that could assist the development of potent and specific in vivo drug candidates.
4-Substituted piperidine-derived trisubstituted ureas are reported as highly potent and selective inhibitors for sEH. The SAR outlines approaches to improve activity against sEH and reduce ion channel and CYP liability. With minimal off-target activity and a good PK profile, the benchmark 2d exhibited remarkable in vitro and ex vivo target engagement. The eutomer entA-2d also elicited vasodilation effect in rat mesenteric artery.
We present the strong fluorescence effect, a new 392 nm emission peak appearing after binding of a naphtol-urea inhibitor XIIa to the enzyme epoxide hydrolase (EH), along with the quenching of the EH tryptophan fluorescence. We have studied the quenching of the 392-nm peak (attributed to XIIa bound inside the active center of the enzyme) of the mixture EH +XIIa by various strong transparent inhibitors (competing with XIIa for binding to EH), and measured the corresponding values of the Stern-Volmer constants, K(mix)(SV). Strong EH inhibitors demonstrate different replacement behavior which can be used to distinguish them. We further demonstrate a novel fluorescent assay which allows to distinguish highly potent inhibitors and to visualize the strongest among them. We generated our assay calibration curve based on the quenching data, by plotting quenching strength K(mix)(SV) versus inhibiting strength, IC(50) values. We used moderate inhibitors for the assay plot generation. We then applied this curve to determine IC(50) values for several highly potent inhibitors, with IC(50) values at the limit of the IC(50) detection sensitivity by colorimetric enzyme assay. IC(50) values determined from our quenching assay show correlation with IC(50) values determined in the literature by more sensitive radioactive-based assay and allow differentiating the inhibitors potency in this group. To our knowledge, this is the first inhibitor assay of such kind. Chemical inhibition of EH is an important technology in the treatment of various cardiovascular diseases, therefore, this tool may play a crucial role in discovering new inhibitor structures for therapeutic EH inhibition.
Spirocyclic secondary amine-derived trisubstituted ureas were identified as highly potent, bioavailable and selective soluble epoxide hydrolase (sEH) inhibitors. Despite good oral exposure and excellent ex vivo target engagement in blood, one such compound, rac-1a, failed to lower blood pressure acutely in spontaneously hypertensive rats (SHRs). This study posed the question as to whether sEH inhibition provides a robust mechanism leading to a significant antihypertensive effect.
We investigated N-adamantyl-N'-phenyl urea derivatives as simple sEH inhibitors. Salicylate ester derivatives have high inhibitory activities against human sEH, while the free benzoic acids are less active. The methyl salicylate derivative is a potent sEH inhibitor, which also has high metabolic and chemical stabilities; suggesting that such inhibitors are potential lead molecule for bioactive compounds acting in vivo.
Background and objective:
COPD is a complex polygenic disease in which gene-environment interactions are very important. The gene encoding microsomal epoxide hydrolase (EPHX1) is one of several candidate loci for COPD pathogenesis and is highly polymorphic. Based chi on the polymorphisms of EPHX1 gene (tyrosine/histidine 113, histidine/arginine 139), the population can be classified into four groups of putative EPHX1 phenotypes (fast, normal, slow and very slow). A number of studies have investigated the association between the genotypes and phenotypes of EPHX1 and COPD susceptibility in different populations, with inconsistent results. A systematic review and meta-analysis of the published data was performed to gain a clearer understanding of this association.
Methods:
The MEDLINE database was searched for case-control studies published from 1966 to August 2007. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated.
Results:
Sixteen eligible studies, comprising 1847 patients with COPD and 2455 controls, were included in the meta-analysis. The pooled result showed that the EPHX1 113 mutant homozygote was significantly associated with an increased risk of COPD (OR 1.59, 95% CI: 1.14-2.21). Subgroup analysis supported the result in the Asian population, but not in the Caucasian population. When the analysis was limited to only the larger-sample-size studies, studies in which controls were in Hardy-Weinberg equilibrium and studies in which controls were smokers/ex-smokers, the pooled results supported the conclusion. The EPHX1 139 heterozygote protected against the development of COPD in the Asian population, but not in the Caucasian population. The other gene types of EPHX1 113 and EPHX1 139 were not associated with an increased risk of COPD. The slow activity phenotype of EPHX1 was associated with an increased risk of COPD. The fast activity phenotype of EPHX1 was a protective factor for developing COPD in the Asian population, but not in the Caucasian population. However, the very slow activity phenotype of EPHX1 was a risk for developing COPD in the Caucasian population, but not in the Asian population.
Conclusions:
The polymorphisms of EPHX1 113 and EPHX1 139 are genetic contributors to COPD susceptibility in Asian populations. The phenotypes of EPHX1 were contributors to overall COPD susceptibility.
A data set of 348 urea-like compounds that inhibit the soluble epoxide hydrolase enzyme in mice and humans is examined. Compounds having IC(50) values ranging from 0.06 to >500 microM (murine) and 0.10 to >500 microM (human) are categorized as active or inactive for classification, while quantitation is performed on smaller compound subsets ranging from 0.07 to 431 microM (murine) and 0.11 to 490 microM (human). Each compound is represented by calculated structural descriptors that encode topological, geometrical, electronic, and polar surface features. Multiple linear regression (MLR) and computational neural networks (CNNs) are employed for quantitative models. Three classification algorithms, k-nearest neighbor (kNN), linear discriminant analysis (LDA), and radial basis function neural networks (RBFNN), are used to categorize compounds as active or inactive based on selected data split points. Quantitative modeling of human enzyme inhibition results in a nonlinear, five-descriptor model with root-mean-square errors (log units of IC(50) [microM]) of 0.616 (r(2) = 0.66), 0.674 (r(2) = 0.61), and 0.914 (r(2) = 0.33) for training, cross-validation, and prediction sets, respectively. The best classification results for human and murine enzyme inhibition are found using kNN. Human classification rates using a seven-descriptor model for training and prediction sets are 89.1% and 91.4%, respectively. Murine classification rates using a five-descriptor model for training and prediction sets are 91.5% and 88.6%, respectively.
Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.
1. Introduction Rat liver epoxide hydratase [EC 4.2.1.631 which catalyses the conversion of epoxides to trurans-dihydro- diols has been purified to apparent homogeneity as determined by three independent criteria [l] . The preparation obtained was capable of catalysing the hydration of both styrene oxide and the 4,5- (K- region)epoxide of benzo(a)pyrene [ 11. Epoxides of polycyclic hydrocarbons have been implicated as the agents responsible for the cytotoxic and carcinogenic properties of such compounds (for reviews see [2-41). A detailed knowledge of the properties of epoxide hydratase may, therefore, contribute towards an understanding of the mechanisms of cytotoxicity and carcinogenesis. We report here the results of initial investigations with the pure enzyme. 2. Materials and methods Anhydrous hydrazine was obtained from Pierce Chem. Co., USA. Carboxypeptidases A and B were isopropylphosphate treated preparations and were obtained from Worthington Biochemical Co. Carboxy- peptidase Y (from Yeast) and Carboxypeptidase P (from Penicillium) were kindly donated by Dr R. Hayashi from Kyoto University, Japan, and Dr E. Ichishima, Tokyo Noko University, Japan. Epoxide hydratase activity was assayed under the conditions 296 described in detail [5], specifically in the absence of Tween 80. 2.1. Amino acid analysis Samples containing 250 ,ug protein were dialysed against 0.2 M borate buffer, pH 9.0, free ammonia was removed by placing samples in a boiling water bath for 5 min and samples were dried in vacua over H2S04. Samples were hydrolysed under reduced pressure at 108 + 1°C [6] in 1 ml of twice distilled 5.7 N HCl for 24 h and 144 h in evacuated sealed tubes. The hydrolysates were dried under vacuum with a rotary evaporator at 80°C. The dried residues were divided into five portions and one portion (50 pg) was applied to one column. The analysis of the hydrolysates was carried out with a Beckman amino acid analyser model 4255, equipped with a 5 cm column (for basic amino acids) and 50 cm column (for acidic and neutral amino acids) at a flow rate of 30 ml/h according to Spackmann et al. [7]. Trypto- phan was analysed using 5% thioglycolic acid [8]. Tyrosine and tryptophan residues were also determined spectrophotometrically in 0.1 N NaOH according to Goodwin Morton [9]. Cyst(e)ine methionine residues were also determined after performic acid oxidation of the protein [lo] N-terminal analysis was performed using [‘“Cl - dinitrofluorobenzene [ 1 l] and the Edman degrada- tion methods [ 121. C-terminal analysis was performed by hydrazinolysis [ 131 and by carboxypeptidase
A molecule isolated from the cerebrospinal fluid of sleep-deprived cats has been chemically characterized and identified as
cis-9,10-octadecenoamide. Other fatty acid primary amides in addition to cis-9,10-octadecenoamide were identified as natural
constituents of the cerebrospinal fluid of cat, rat, and human, indicating that these compounds compose a distinct family
of brain lipids. Synthetic cis-9,10-octadecenoamide induced physiological sleep when injected into rats. Together, these results
suggest that fatty acid primary amides may represent a previously unrecognized class of biological signaling molecules.
Bile acids, such as taurocholate, have been shown to be transported into hepatocyte smooth endoplasmic reticulum (SER) vesicles. This process is Na(+)-independent, electrogenic, inhibitable by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and taurochenodeoxycholate, with a Km of 352 microM and a Vmax of 29.6 nmol/mg protein/min. The observed transport is mediated by the bifunctional protein, microsomal epoxide hydrolase (mEH) which can also mediate bile acid transport into hepatocytes across the sinusoidal plasma membrane (von Dippe, P., Amoui, M., Alves, C., and Levy, D. (1993) Am. J. Physiol. 264, G528-G534). mEH was isolated from SER membranes by immunoprecipitation with monoclonal antibody (mAb) 25D-1 which recognizes this protein on the surface of intact hepatocytes. The SER-derived protein exhibited an apparent molecular weight, isoelectric point, N-terminal amino acid sequence, and mEH-specific activity that were indistinguishable from the plasma membrane form of the enzyme. Proteoliposome reconstitution of the SER taurocholate transport system indicated that mEH was absolutely required for the expression of transport capacity. The interaction of mAb 25D-1 with mEH on intact right-side-out SER vesicles demonstrated that the epitope found on the surface of hepatocytes was also found on the cytoplasmic surface of these vesicles (80%) and in the lumen (20%) suggesting the presence of two forms of this protein in the SER, the latter from being sorted to the cell surface. The existence of two orientations of this protein in the SER was confirmed by the sensitivity to tryptic digestion, where 75% of the mAb epitope was accessible to the enzyme. The loss of the 25D-1 epitope correlated with loss of taurocholate transport capacity. The role of mEH in the transport process and the orientation of the transporting isoform was further established by demonstrating that mAb 25A-3, which also reacts with mEH on the hepatocyte surface, was able to directly inhibit taurocholate transport in the SER vesicle system. These and previous results thus establish that isoforms of mEH can mediate taurocholate transport at the sinusoidal plasma membrane and in SER vesicles and that this bifunctional protein can exist in two orientations in the SER membrane. The association of bile acids with the SER suggests a possible role of intracellular vesicles in the transhepatocellular movement of bile acids from the sinusoidal to the canalicular compartment.
Previous studies have suggested that the enzyme microsomal epoxide hydrolase (mEH) is able to mediate sodium-dependent transport of bile acids such as taurocholate into hepatocytes (von Dippe, P., Amoui, M., Alves, C., and Levy, D.(1993) Am. J. Physiol. 264, G528-G534). In order to characterize directly the putative transport properties of the enzyme, a pCB6 vector containing the cDNA for this protein (pCB6-mEH) was transfected into Madin-Darby canine kidney (MDCK) cells, and stable transformants were isolated that could express mEH at levels comparable with the levels expressed in hepatocytes. Sodium-dependent transport of taurocholate was shown to be dependent on the expression of mEH and to be inhibited by the bile acid transport inhibitor 4,4'-diisothiocyanostilbene-2,2'disulfonic acid (DIDS), as well as by other bile acids. Kinetic analysis of this system indicated a Km of 26.3 microM and a Vmax of 117 pmol/mg protein/min. The Km value is essentially the same as that observed in intact hepatocytes. The transfected MDCK cells also exhibited sodium-dependent transport of cholate at levels 150% of taurocholate in contrast to hepatocytes where cholate transport is only 30% of taurocholate levels, suggesting that total hepatocyte bile acid transport is a function of multiple transport systems with different substrate specificities, where mEH preferentially transports cholate. This hypothesis is further supported by the observation that a monoclonal antibody that partially protects (26%) taurocholate transport from inhibition by DIDS in hepatocytes provides almost complete protection (88%) from DIDS inhibition of hepatocyte cholate transport, suggesting that taurocholate is also taken up by an alternative system not recognized by this antibody. Additional support for this concept is provided by the observation that the taurocholate transport system is almost completely protected (92%) from DIDS inhibition by this antibody in MDCK cells that express mEH as the only bile acid transporter. These results demonstrate that mEH is expressed on the surface of hepatocytes as well as on transfected MDCK cells and is able to mediate sodium-dependent transport of taurocholate and cholate.
A tritiated photoaffinity labelling analogue of tamoxifen, [(2-azido-4-benzyl)-phenoxy]-N-ethylmorpholine (azido-MBPE), was used to identify the anti-oestrogen-binding site (AEBS) in rat liver tissue [Poirot, Chailleux, Fargin, Bayard and Faye (1990) J. Biol. Chem. 265, 17039-17043]. UV irradiation of rat liver microsomal proteins incubated with tritiated azido-MBPE led to the characterization of two photolabelled proteins of molecular masses 40 and 50 kDa. The amino acid sequences of proteolytic products from the 50 kDa protein were identical with those from rat microsomal epoxide hydrolase (mEH). Treatment of hepatocytes with anti-sense mRNA directed against mEH abolished AEBS in these cells. In addition we found that tamoxifen and N-morpholino-2-[4-(phenylmethyl)phenoxy]ethanamine, a selective ligand of AEBS, were potent inhibitors of the catalytic hydration of styrene oxide by mEH. However, functional overexpression of the human mEH did not significantly modify the binding capacity of [3H]tamoxifen. Taken together, these results suggest that the 50 kDa protein, mEH, is necessary but not sufficient to reconstitute AEBS.
Microsomal epoxide hydrolase (mEH) is involved in the metabolism of tobacco-derived carcinogens. Polymorphisms in exons 3 and 4 of the EPHX gene have been reported to be associated with variations in mEH activity. We examined whether the predicted mEH activity modified the lung cancer risk among 150 cases and 172 controls, all French Caucasian smokers. A significant association was found between predicted mEH activity and lung cancer (P < 0.02), with a dose-effect relationship (P < 0.005). The risks associated with intermediate and high activities, compared to low activity, were 1.65 (95% CI, 0.95-2.86) and 2.66 (95% CI, 1.33-5.33), respectively. The effect of mEH activity on lung cancer risk was not significantly modified by smoking exposure, CYP1A1 genotype, or GSTM1 genotype. mEH may thus be an important genetic determinant of smoking-induced lung cancer.
Inhibition of xenobiotic-metabolizing enzymes by metals may represent an important mechanism in regulating enzyme activity. Fourteen cations were evaluated for inhibition of microsomal epoxide hydrolase (mEH) (mouse, rat, and human liver), soluble epoxide hydrolase (sEH) (mouse, rat, and human liver), and recombinant potato sEH. Of the metals tested, Hg2+ and Zn2+ were the strongest inhibitors of mEH, while Cd2+ and Cu2+ were also strong inhibitors of sEH (I50 for all approximately 20 microM). Nickel (divalent) and Pb2+ were moderate inhibitors, but Al2+, Ba2+, Ca2+, Co2+, Fe2+, Fe3+, Mg2+, and Mn2+ were weak inhibitors of both mEH and sEH (less than 50% inhibition by 1 mM metal). Six anions (acetate, bromide, chloride, nitrate, perchlorate, and sulfate) were tested and found to have no effect on the inhibition of sEH or mEH by cations. The kinetics and type of inhibition for zinc inhibition of sEH and mEH were examined for mouse, rat, human, and potato. Zinc inhibits mEH in a competitive manner. Inhibition of human and potato sEH was noncompetitive, but interestingly, zinc inhibition of mouse sEH was very strong and uncompetitive. Inhibition by zinc could be reversed by adding EDTA to the incubation buffer. Additionally, mouse liver microsomes and cytosol were incubated with these chelators. Following incubation at 4 degrees C, samples were dialyzed to remove chelator. Both mEH and sEH activity recovered was greater in samples treated with chelator than in control incubations. Similar treatment with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) did not affect enzyme activity recovered. During systemic inflammation, hepatic metallothionien is induced, and liver metal concentrations increase while serum metal concentrations are decreased. The inhibition of microsomal and soluble epoxide hydrolase by metals may represent a mechanism of down-regulation of enzyme activity during inflammation.
The structures of two alkylurea inhibitors complexed with murine soluble epoxide hydrolase have been determined by x-ray crystallographic methods. The alkyl substituents of each inhibitor make extensive hydrophobic contacts in the soluble epoxide hydrolase active site, and each urea carbonyl oxygen accepts hydrogen bonds from the phenolic hydroxyl groups of Tyr(381) and Tyr(465). These hydrogen bond interactions suggest that Tyr(381) and/or Tyr(465) are general acid catalysts that facilitate epoxide ring opening in the first step of the hydrolysis reaction; Tyr(465) is highly conserved among all epoxide hydrolases, and Tyr(381) is conserved among the soluble epoxide hydrolases. In one enzyme-inhibitor complex, the urea carbonyl oxygen additionally interacts with Gln(382). If a comparable interaction occurs in catalysis, then Gln(382) may provide electrostatic stabilization of partial negative charge on the epoxide oxygen. The carboxylate side chain of Asp(333) accepts a hydrogen bond from one of the urea NH groups in each enzyme-inhibitor complex. Because Asp(333) is the catalytic nucleophile, its interaction with the partial positive charge on the urea NH group mimics its approach toward the partial positive charge on the electrophilic carbon of an epoxide substrate. Accordingly, alkylurea inhibitors mimic features encountered in the reaction coordinate of epoxide ring opening, and a structure-based mechanism is proposed for leukotoxin epoxide hydrolysis.
Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr(465) and Tyr(381) act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr(465) and Tyr(381) are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr(465) and Tyr(381) decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr(465) is conserved in microsomal EHs. The substitution of analogous Tyr(374) with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.
The soluble epoxide hydrolase (sEH) plays a significant role in the biosynthesis of inflammation mediators as well as xenobiotic
transformations. Herein, we report the discovery of substituted ureas and carbamates as potent inhibitors of sEH. Some of
these selective, competitive tight-binding inhibitors with nanomolar Ki values interacted stoichiometrically with the homogenous recombinant murine and human sEHs. These inhibitors enhance cytotoxicity
of trans-stilbene oxide, which is active as the epoxide, but reduce cytotoxicity of leukotoxin, which is activated by epoxide hydrolase
to its toxic diol. They also reduce toxicity of leukotoxin in vivo in mice and prevent symptoms suggestive of acute respiratory distress syndrome. These potent inhibitors may be valuable tools
for testing hypotheses of involvement of diol and epoxide lipids in chemical mediation in vitro or in vivo systems.
On the basis of drug interactions with carbamazepine epoxide, it has been hypothesized that valproic acid and valpromide are inhibitors of epoxide hydrolase, but the role of epoxide hydrolase in these interactions has not been clearly established. In this study, therapeutic concentrations of valproic acid (<1 mmol/L) and valpromide (<10 mol/L) inhibited hydrolysis of carbamazepine epoxide and styrene oxide in human liver microsomes and in preparations of purified human liver microsomal epoxide hydrolase. Valpromide (K1 = 5 mol/L) was 100 times more potent than valproic acid (K1 = 550 mol/L) as an inhibitor of carbamazepine epoxide hydrolysis in microsomes. After administration of carbamazepine epoxide to volunteers, the transdihydrodiol formation clearance was decreased 20% by valproic acid (blood concentration ≈ 113 mol/L) and 67% by valpromide (blood concentration < 10 mol/L). For both valproic acid and valpromide, a striking similarity exists between in vitro and in vivo inhibitory potencies. Valproic acid and valpromide are the first drugs known to inhibit microsomal epoxide hydrolase, an important detoxification enzyme, at therapeutic concentrations.
Purpose. The purpose of this study was to evaluate the in vitro inhibitory potency of various amide analogues and derivatives of valproicacid toward human microsomal epoxide hydrolase (mEH).
Methods. mEH inhibition was evaluated in human liver microsomeswith 25 (S)-(+)-styrene oxide as the substrate. Inhibitory potencyexpressed as the median inhibitory concentration (IC50) was calculatedfrom the formation rate of the enzymatic product,(S)-(+)-1-phenyl-1,2-ethanediol.
Results. Inhibitory potency was directly correlated with lipophilicityand became significant for amides with a minimum of eight carbonatoms. Branched eight-carbon amides were more potent inhibitors thantheir straight chain isomer, octanamide. N-substituted valproylamideanalogues had reduced or abolished inhibition potency with theexception of valproyl hydroxamic acid being a potent inhibitor. Inhibitionpotency was not stereoselective in two cases of chiral valpromideisomers. Valproyl glycinamide, a new antiepileptic drug currentlyundergoing phase II clinical trials and its major metabolite valproylglycine were weak mEH inhibitors. Acid isomers of valproic acid werenot potent mEH inhibitors.
Conclusions. The structural requirements for valproylamide analoguesfor potent in vitro mEH inhibition are: an unsubstituted amide moiety;two saturated alkyl side chains; a minimum of eight carbons in themolecule.
The cDNA of the microsomal Juvenile Hormone Epoxide Hydrolase (JHEH) from Manduca sexta was expressed in vitro in the baculovirus system. In insect cell culture, the recombinant enzyme (Ms-JHEH) was produced at a high level (100 fold over background EH catalytic activity). As expected, Ms-JHEH was localized in the microsomal fraction with a molecular mass of approximately 50 kDa. Ms-JHEH showed a substrate and inhibitor spectrum similar to the wild type JHEH isolated from eggs of M. sexta. Its enzymatic activity was the highest for Juvenile Hormone III. Ms-JHEH hydrolyzed several trans-epoxides faster than cis-epoxides. A putative hydroxyl-acyl enzyme intermediate was isolated suggesting a catalytic mechanism of Ms-JHEH similar to the mammalian EHs.
Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and that has been shown to effect serotonergic receptor responses and block gap junction communication. Herein, the potentiation of the 5-HT1A receptor response is disclosed, and a study of the structural features of oleamide required for potentiation of the 5-HT2A and 5-HT1A response to serotonin (5-HT) is described. Of the naturally occurring fatty acids, the primary amide of oleic acid (oleamide) is the most effective at potentiating the 5-HT2A receptor response. The structural features required for activity were found to be highly selective. The presence, position, and stereochemistry of the delta9-cis double bond is required, and even subtle structural variations reduce or eliminate activity. Secondary or tertiary amides may replace the primary amide but follow a well defined relationship requiring small amide substituents, suggesting that the carboxamide serves as a hydrogen bond acceptor but not donor. Alternative modifications at the carboxamide as well as modifications of the methyl terminus or the hydrocarbon region spanning the carboxamide and double bond typically eliminate activity. A less extensive study of the 5-HT1A potentiation revealed that it is more tolerant and accommodates a wider range of structural modifications. An interesting set of analogs was identified that inhibit rather than potentiate the 5-HT2A, but not the 5-HT1A, receptor response, further suggesting that such analogs may permit the selective modulation of serotonin receptor subtypes and even have opposing effects on the different subtypes.
Six different 1,2-epoxycycloalkanes, whose rings were constituted of 5 to 12 carbon atoms, were tested as possible inhibitors of epoxide-metabolizing enzymes and substrates for the microsomal and cytosolic epoxide hydrolases (mEH, cEH) in mouse liver. The geometric configurations and the relative steric hindrances of these epoxides were estimated from their ease of hydrolysis in acidic conditions to the corresponding diols, their abilities to react with nitrobenzylpyridine, and the chemical shifts of the groups associated with the oxirane rings measured by proton and 13C-NMR. The cyclopentene, -hexene, -heptene, -octene and -decene oxides adopted mainly a cis-configuration. By contrast, cyclododecene oxide presented a trans-configuration. Steric hindrance increased with the size of the ring and was particularly strong when cyclooctene, -decene and -dodecene oxides were considered. With the exception of cyclohexene oxide, all the compounds were weak inhibitors of EH and glutathione S-transferase (GST) activities. Cyclohexene oxide exhibited a selective inhibition of the mEH with an I50 of 4.0.10(-6) M. As the size of the ring increased, inhibitory potency was gradually lost. The cEH and the GST activities were less sensitive to the inhibitory effects of these epoxides (I50, 1 mM or above). A marked difference between the substrate selectivities of mEH and cEH for these epoxides was observed. The mEH hydrated all of the cyclic epoxides, although some of them at a very low rate; the best substrate was the cycloheptene oxide (2.3 nmol/min/mg protein). On the other hand, cyclodecene oxide was a substrate of cEH, but no diol formation was detected when cyclopentene, -hexene and -dodecene oxides were incubated with cytosolic enzyme.
1. Several aromatic and olefinic compounds are converted to intermediate arene and alkene oxides by mammalian mono-oxygenases. Intermediate arene oxides rearrange non-enzymically to phenols. Arene and alkene oxides are converted by epoxide hydrases to vicinal diols and by glutathione S-epoxide conjugases to glutathione conjugates. Due to their high electrophilic reactivity, such oxiranes also bind to proteins, RNA and DNA. Mutagenic, carcinogenic and cytotoxic effects of several aromatic and olefinic compounds appear to be due to the formation of intermediate epoxides and their reaction with tissue constituents. Whether a given aromatic or olefinic compound produces such an effect would thus depend on a variety of factors, such as the relative rate of formation and degradation of the intermediate oxirane, on its stability with respect to spontaneous isomerization to the corresponding phenol and on its chemical electrophilic reactivity. 2. Epoxide hydrases, which convert such intermediate oxiranes to much less reactive vicinal diols, have been studied in greater detail. Epoxide hydrase activity is found in mouse, rat, guinea-pig, rabbit, pig, Rhesus monkey and human liver. Activity is high in liver, low in kidney, very low in intestine and lung and not detectable in muscle, spleen, heart and brain. The enzyme is located exclusively in microsomal membranes. Epoxide hydrase activity is markedly increased after pretreatment of rats with phenobarbital or 3-methyl-cholanthrene and during maturation of rats. These increases are reminiscent of similar increases in microsomal mono-oxygenases. However, the extents of induction of total levels of these two enzymes or enzyme families are not comparable and are under separate genetic control. 3. Several stereochemical properties of the reaction catalysed by epoxide hydrases have been studied with microsomal preparations. With styrene oxide and naphthalene oxide as substrates, attack by H218O occurs virtually exclusively at the 2-position. Product glycols which are stereochemically fixed by a ring structure invariably have the trans-configuration. Hydration of some acyclic alkene oxides has also been found to proceed via a trans-opening of the oxirane-ring. Cyclohexene oxide, benzene oxide and naphthalene 1,2-oxide are converted predominantly to 1R,2R-trans-diols, while in the case of phenanthrene 9,10-oxide the 1S,2S,-trans-diol predominates. 4. Epoxide hydrase from guinea-pig liver microsomes was solubilized and purified, based on an assay with styrene oxide as substrate. The specific activity after the last purification step is about 40 times higher than in the crude homogenate. This increase is not due to the removal of an inhibitor. About 30 % of the activity of the purified preparation is lost within 1-2 days. However, the remaining activity is remarkably stable. Gel electrophoresis of the final (stable) preparation shows one major band corresponding to a mol. wt. of approx. 50 000. However, several minor bands are also present. 5. Several properties of epoxide hydrase were investigated with this purified preparation. While no clearcut pH optimum could be observed with microsomal preparations (broad 'optimum' between 7 and 9) a sharp pH profile was obtained with the purified preparation with its optimum at pH 9. Non-enzymic hydration was significant (>5% only below pH 6·5. The KM with respect to styrene oxide as substrate is 2-8 × 10-4M and the apparent Vmax. 2-4 μmol product/mg N per 5 min. No metal ions or other low mol. wt. co-factors are necessary for maximal activity. High concentrations of substrate inhibit the enzyme, whereas product diols have no effect. Several inhibitors of drug-metabolizing enzymes (SKF 525-A), piperonyl butoxide, αnaphthoflavone) do not influence epoxide hydrase activity, while sulphydryl reagents slightly, but significantly, inhibit the enzyme. Several alcohols, ketones and imidazoles stimulate the enzyme. Kinetic analysis of the activation by the potent stimulator, metyrapone, indicates negative co-operativity with the substrate. 6. The active site of the enzyme readily accommodates, as substrates or competitive inhibitors, monosubstituted oxiranes with a lipophilic substituent larger than an ethyl group, suggesting hydrophobic binding sites near the active site. With oxiranes having such a lipophilic substituent, the enzyme interacts with mono-, 1,1-di- and cis-1,2-disubstituted oxiranes, but not with trans-1,2-disubstituted oxiranes or tri- or tetra-substituted oxiranes, suggesting that increasing bulk around the oxirane ring prevents the approach of the oxirane to the active site. Several oxiranes fused to alicyclic rings (cyclohexene oxide, 1,2,3,4-tetrahydronaphthalene 1,2-epoxide) are potent inhibitors but very poor substrates. Kinetic analysis revealed non-competitive inhibition with respect to the substrate, styrene oxide. Styrene sulphide (an analogue of the competitive inhibitor, styrene oxide) but not cyclohexene sulphide (an analogue of the non-competitive inhibitor, cyclohexene oxide) has inhibitory activity, suggesting differing structural requirements for the sites involved in competitive and non-competitive inhibition. The most potent inhibitor discovered so far is 1,1,1-trichloropropene 2,3-oxide, which is on the other hand a poor substrate. Inhibition by this compound is of the un-competitive type. Structure-activity relationship for substrates and inhibitors of purified human epoxide hydrase are qualitatively identical to the ones discussed above for the purified guinea-pig enzyme. 7. Evidence suggesting the presence of more than one liver enzyme capable of hydrating epoxides include differential stabilities, different ratios of hydrase activity towards various epoxides in preparations from different species, a different purification factor (activity of the purified preparation compared to liver homogenates) towards benzene oxide as compared to several other epoxides, inability to inhibit hydration of styrene oxide (in purified or particulate preparations) with much higher concentrations of benzene oxide. 8. Evidence indicating the presence of a coupled mono-oxygenase-epoxide hydrase multienzyme complex include the following observations. Substantial amounts of dihydrodiols in the urine of animals treated with aromatic hydrocarbons, despite the high instability of intermediate arene oxides, lack of equilibration between pools of naphthalene oxide formed in situ and of exogenous naphthalene oxide, differential inhibition of 'free epoxide hydrases' at concentrations of 1,1,1-trichloropropene 2,3-oxide which do not affect the 'coupled mono-oxygenase-epoxide hydrase system' selective induction of the coupled system by 3-methylcholanthrene, and high epoxide hydrase activities in solubilized and purified cytochrome P-450 and P-448 fractions, where other microsomal enzymes (glucose-6-phosphatase, cytochrome c reductase) were absent. Such a coupled mono-oxygenase-epoxide hydrase system may be of great relevance to problems such as carcinogenic properties of arene oxides derived from several polycyclic hydrocarbons, and hepatotoxicity of intermediate arene oxides derived from halobenzenes, by circumventing these adverse effects by their rapid conversion to dihydrodiols. Indeed, pretreatment of rats with 3-methylcholanthrene, which selectively induces this coupled system, provides protection from chlorobenzene-evoked hepatotoxicity, whereas pre-treatment with phenobarbital increases the toxic effect, although it induces the total level of epoxide hydrase to a much greater extent than 3-methylcholanthrene. © 1973 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
Epoxide hydrase activity, measured with [3H]styrene oxide as substrate, is present in mammalian liver, kidney, lung, intestine and skin. The hepatic level of the enzyme, measured in vitro with [3H]styrene oxide, benzene oxide or naphthalene-1,2-oxide, is elevated substantially by pretreatment of rats with phenobarbital and to a lesser extent by pretreatment with 3-methylcholanthrene. Metyrapone and 1-(2-isopropylphenyl)-imidazole, two monooxygenase inhibitors, activate epoxide hydrase in vitro, but have no demonstrable effect on the enzyme in vivo. 3,3,3-Trichloropropene oxide, a potent in vitro inhibitor of epoxide hydrase, has no effect on monooxygenase activity measured in vitro with [3H]benzenesulfonanilide. Trichloropropene oxide is extremely toxic. In sub-lethal dosages, it does not significantly inhibit epoxide hydrase activity in vivo, although it and several other epoxides do react with and thereby reduce hepatic levels of glutathione. Cyclohexane oxide, another potent in vitro inhibitor of epoxide hydrase, reduces hepatic glutathione levels to 10% of control values. This relatively non-toxic substance should potentiate the hepatotoxicity of chlorobenzene by inhibiting further metabolism of the toxic chlorobenzene oxide intermediate through either hydration or conjugation with glutathione. Instead, co-administration of cyclohexene oxide and chlorobenzene significantly reduces the rate of metabolism of [14C]chlorobenzene and prevents the hepatic centrilobular necrosis caused by chlorobenzene in rats. Arene oxide-mediated hepatotoxicity apparently is dependent upon a variety of factors including both rates of formation and degradation of arene oxides in tissue. The presently known hydrase inhibitors are not sufficiently selective in their effects on liver cells to permit a quantitative assessment of the relative importance of these factors.
An epoxide hydrase preparation solubilized and partially purified from guinea pig liver microsomes catalyzes the hydration of a variety of epoxides to corresponding glycols. The activity of various epoxides as substrates or as inhibitors of this enzyme is dependent on the nature and stereochemistry of substituents on the oxirane ring. Oxiranes with a 1-aryl substituent (styrene oxides) or with certain 1-alkyl substituents (1-octene oxide, phenyl 2,3-epoxypropyl ethers) are among the best substrates for the enzyme and are competitive inhibitors for hydration of styrene-t oxide. 1,1-Disubstituted and cis-1,2-disubstituted oxiranes are less active as substrates and inhibitors. Trans-1,2-disubstituted, trisubstituted, and tetrasubstituted oxiranes are virtually inactive as substrates or inhibitors. Certain alicyclic oxiranes such as 1,2-epoxy1,2,3,4-tetrahydronaphthalene and cyclohexene oxide are relatively inactive as substrates but very effective as inhibitors. Inhibition by these compounds appears to be noncompetitive with respect to substrate. 1,1,1-Trichloropropene 2,3-oxide is a very potent uncompetitive inhibitor of epoxide hydrase. Various analogs of epoxides such as azaridines, thiiranes, and oxaziridines, either do not inhibit the enzyme or are relatively weak inhibitors. Alcohols and certain ketones, such as metyrapone, activate the enzyme. Structure-activity correlations with the epoxide hydrase are distinctly different from those of squalene oxidocyclase.
The specific activities of microsomal epoxide hydrolase with 16 alpha, 17 alpha-epoxyandrosten-3-one (androstene oxide) as substrate were measured in various metabolically important and in various steroidogenic organs of the male and female rat and compared with the activities of 16 alpha, 17 alpha-epoxyestratrienol (estroxide) and benzo[a]pyrene 4,5-oxide. Androstene oxide was an exceptionally good substrate. The specific activities differed widely between organs but the ratio of the activities towards these substrates was constant in all organs investigated. The ratios compared to benzo[a]pyrene 4,5-oxide were 2.5 for estroxide, and 8.6 for androstene oxide. The ontogenetic development of specific epoxide hydrolase activity in the livers of both sexes reached a maximum at about day 40 and descended to the adult enzyme level at about 45 days in males and clearly later in females. While in the livers and ovaries significant increases of the enzyme activity with increasing age took place before day 28, the specific activity remained very low in the testis until day 28 and then rose suddenly. During all these differential developments no significant changes in the ratios of activities towards the three substrates were observed. The specific activity of epoxide hydrolase towards these substrates in subcellular fractions of the rat liver was smooth endoplasmic reticulum greater than microsomes approximately equal to rough endoplasmic reticulum much greater than mitochondria, no activity was detectable in cytosol. The ratio of the activities in the different fractions was similar when measured with androstene oxide, estroxide and styrene oxide as substrates. Microsomal hydrolysis responded to pretreatment of animals with phenobarbital, 3-methylcholanthrene. Arochlor 1254 and trans-stilbene oxide in a manner which was characteristically different for the various agents but similar for the three substrates. Microsomal epoxide hydrolase which was purified to apparent homogeneity was able to hydrolyse the steroid epoxides, but the apparent purification factors were different for the different substrates: 77 for styrene oxide, 45 for estroxide, and 10 for androstene oxide. The three substrates mutually inhibited their hydrolysis by the microsomal fraction. Some differences in the extent of their effect and in the inhibition of the activities by known epoxide hydrolase inhibitors were observed. Similarly, hydrolysis of the steroid epoxides but not of styrene oxide was inhibited by nonionic detergents (Cutscum, Triton X-100 and Emulgen 911). These differences could be due to the presence of different enzymes or a single enzyme, the conformational requirements of which are much more demanding for steroid epoxides than for xenobiotic epoxides. Mono-specific antiserum precipitated epoxide hydrolase activity from solubilized microsomes with dose-response curves which were not distinguishable for androstene oxide, estroxide, benzo[a]pyrene 4,5-oxide and styrene oxide as substrates...
A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or more cloned enzymes. The ability of these enzymes to prevent or enhance the toxicity of drugs and xenobiotics is then measured in situ. Initial parameters for the system were developed and optimized using baculoviruses engineered for expression of the mouse soluble epoxide hydrolase (msEH, EC 3.3.2.3) or the rat cytochrome P4501A1. SF-21 cells expressing msEH were resistant to trans-stilbene oxide toxicity as well as several other toxic epoxides including: cis-stilbene oxide, 1,2,7,8-diepoxyoctane, allylbenzene oxide, and estragole oxide. The msEH markedly reduced DNA and protein adduct formation in SF-21 cells exposed to [3H]allylbenzene oxide or [3H]estragole oxide. On the other hand, 9,10-epoxyoctadecanoic acid and methyl 9,10-epoxyoctadecanoate were toxic only to cells expressing sEH, suggesting that the corresponding fatty acid diols were cytotoxic. This was confirmed by showing that chemically synthesized diols of these fatty acid epoxides were toxic to control SF-21 cells at the same concentration as were the epoxides to cells expressing sEH. A recombinant baculovirus containing a chimeric cDNA formed between the rat P4501A1 and the yeast NADPH-P450 reductase was also constructed and expressed in this system. A model compound, naphthalene, was toxic to SF-21 infected with the rat P4501A1/reductase chimeric co-infecting SF-21 cells with either a human or a rat microsomal EH virus along with P4501A1/reductase virus. These results demonstrate the usefulness of this new system for experimentally analyzing the role of enzymes hypothesized to metabolize endogenous and exogenous chemicals of human health concern.
Human glutathione transferases (GSTs) are a multigene family of enzymes that are involved in the metabolism of a wide range of electrophilic compounds of both exogenous and endogenous origin. GSTs are generally recognized as detoxifying enzymes by catalyzing the conjugation of these compounds with glutathione, but they may also be involved in activation of some carcinogens. The memmalian GSTs can be differentiated into four classes of cytosolic enzymes and two membrane bound enzymes. Human epoxide hydrolases (EHs) catalyze the addition of water to epoxides to form the corresponding dihydrodiol. The enzymatic hydration is essentially irreversible and produces mainly metabolites of lower reactivity that can be conjugated and excreted. The reaction of EHs is therefore generally regarded as detoxifying. The mammalian EHs can be distinguished by their physical and enzymatic properties. Microsomal EH (mEH) exhibits a broad substrate specificity, while the soluble EH (sEH) is an enzyme with a "complementary" substrate specificity to mEH. Cholesterol EH and leukotriene A4 hydrolase are two EHs with very limited substrate specificity. The activities of either GSTs or EHs expressed in vivo exhibit a relatively large interindividual variation, which might be explained by induction, inhibition, or genetic factors. These variations in levels or activities of individual isoenzymes are of importance with respect to an individual's susceptibility to genotoxic effects. This article gives a general overview of GSTs and EHs, discussing the modulation of activities, determination of these enzymes ex vivo, and the polymorphic expression of some isoenzymes.
The first-pass metabolism of foreign compounds in the lung is an important protective mechanism against oxidative stress. We investigated whether polymorphisms in the gene for microsomal epoxide hydrolase (mEPHX), an enzyme involved in this protective process, had any bearing on individual susceptibility to the development of chronic obstructive pulmonary disease (COPD) and emphysema.
We designed PCR-based genotyping assays to detect variant forms of mEPHX that confer slow and fast activity. We used these assays to screen 203 blood-donor controls and groups of patients with asthma (n = 57), lung cancer (n = 50), COPD (n = 68), and emphysema (n = 94), who were attending specialised clinics in Edinburgh, UK.
The proportion of individuals with innate slow mEPHX activity (homozygotes) was significantly higher in both the COPD group and the emphysema group than in the control group (COPD 13 [19%] vs control 13 [6%]; emphysema 21 [22%] vs 13 [6%]). The odds ratios for homozygous slow activity versus all other phenotypes were 4.1 (95% CI 1.8-9.7) for COPD and 5.0 (2.3-10.9) for emphysema.
Genetic polymorphisms in xenobiotic enzymes may have a role in individual susceptibility to oxidant-related lung disease. Epoxide derivatives of cigarette-smoke components may be the cause of some of the lung damage characteristic of these diseases.
Naphthalene-induced Clara cell toxicity in the mouse is associated with the covalent binding of electrophilic metabolites to cellular proteins. Epoxide and quinone metabolites of naphthalene are proposed to be the reactive metabolites responsible for covalent binding to proteins. To identify the nature of reactive metabolites bound to proteins (cysteine residues), we alkaline-permethylated proteins obtained from mouse Clara cells incubated with 0.5 mM naphthalene in vitro. Alkaline permethylation of protein adducts produced (methylthio)naphthalene derivatives detected by GC-MS. 3,4-Dimethoxy(methylthio)naphthalene was observed to be a predominant (methylthio)naphthalene derivative formed in the alkaline-permethylated protein sample obtained from Clara cells after exposure to naphthalene. This indicates that 1,2-naphthoquinone is a major metabolite covalently bound to cysteine residues of the cellular proteins. We have developed an immunoblotting approach to detect 1,2-naphthoquinone covalently bound to cysteine residues of proteins [Zheng, J., and Hammock, B. D. (1996) Chem. Res. Toxicol. 9, 904-909]. To identify 1,2-naphthoquinone covalently bound to sulfur nucleophiles of proteins, homogenates obtained from naphthalene-exposed Clara cells were separated by SDS-PAGE followed by Western blotting and immunostaining with the antibodies. Two protein bands with 24 and 25 kDa were detected by the antibodies, further supporting the view that 1,2-naphthoquinone is a reactive metabolite of naphthalene which binds to Clara cell proteins in vitro.
Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and has been shown to effect serotonergic systems and block gap junction communication in a structurally specific manner. Herein, the structural features of oleamide required for inhibition of the gap junction-mediated chemical and electrical transmission in rat glial cells are defined. The effective inhibitors fall into two classes of fatty acid primary amides of which oleamide and arachidonamide are the prototypical members. Of these two, oleamide constitutes the most effective, and its structural requirements for inhibition of the gap junction are well defined. It requires a chain length of 16-24 carbons of which 16-18 carbons appears optimal, a polarized terminal carbonyl group capable of accepting but not necessarily donating a hydrogen bond, a Delta9 cis double bond, and a hydrophobic methyl terminus. Within these constraints, a range of modifications are possible, many of which may be expected to improve in vivo properties. A select set of agents has been identified that serves both as oleamide agonists and as inhibitors of fatty acid amide hydrolase, which is responsible for the rapid inactivation of oleamide.
The membrane-bound form of epoxide hydrolase and NADPH-cytochrome P-450 (c) reductase are two important enzymes involved in the bioactivation/bioinactivation balance of cerebral tissue. In vivo, the developmental profiles and regional localizations of these two enzymes were investigated in the rat. The regional distribution study showed that they are ubiquitously present among the major brain structures. Both enzyme activities were present in the brain prior to birth, and hence tissue from early developmental stages is suitable to develop in vitro cellular or organotypic models for toxicity studies involving these metabolic pathways. Because various neurotoxicological effects can be dependent on spatio-temporally regulated cell-cell interactions, we aimed to employ organotypic tissue cultures in which the cytoarchitecture was well preserved. In such cultures, the temporal expression profiles of epoxide hydrolase and NADPH cytochrome(c) P-450 reductase reflected the in vivo situation. The technically less demanding pure neuronal and glial cell cultures were also investigated. Detoxification of benzopyrene-4,5-epoxide and superoxide production arising from the reductive metabolism of various drugs were determined in all three systems. The results indicate that though organotypic culture is a good model for the metabolic pathways studied, less complicated single cell cultures can also represent appropriate model systems, providing that the expression of the enzymes involved has been first established in these systems. NADPH-cytochrome P-450 reductase-dependent metabolism is active in both neuronal and glial cells, whereas the detoxification of reactive epoxides occurs mainly in glia.
The potent analgesic effects of cannabis-like drugs and the presence of CB1-type cannabinoid receptors in pain-processing areas of the brain and spinal cord indicate that endogenous cannabinoids such as anandamide may contribute to the control of pain transmission within the central nervous system (CNS). Here we show that anandamide attenuates the pain behaviour produced by chemical damage to cutaneous tissue by interacting with CB1-like cannabinoid receptors located outside the CNS. Palmitylethanolamide (PEA), which is released together with anandamide from a common phospholipid precursor, exerts a similar effect by activating peripheral CB2-like receptors. When administered together, the two compounds act synergistically, reducing pain responses 100-fold more potently than does each compound alone. Gas-chromatography/mass-spectrometry measurements indicate that the levels of anandamide and PEA in the skin are enough to cause a tonic activation of local cannabinoid receptors. In agreement with this possibility, the CB1 antagonist SR141716A and the CB2 antagonist SR144528 prolong and enhance the pain behaviour produced by tissue damage. These results indicate that peripheral CB1-like and CB2-like receptors participate in the intrinsic control of pain initiation and that locally generated anandamide and PEA may mediate this effect.
We conducted a nested case-control study to examine the association of microsomal epoxide hydrolase polymorphism with spontaneous abortion. The analysis included 127 cases and 107 controls from a rural community in China. The prevalence of the homozygous wild-type (AA), the heterozygous variant (Aa), and the homozygous variant (aa) in exon 3 of epoxide hydrolase was 13.4%, 34.6%, and 52.0% in cases and 27.1%, 30.8%, and 42.1% in controls, respectively. In contrast, the variant genotypes in exon 4 of epoxide hydrolase were less frequent in cases than controls. Using women with genotype AA as the referent, the adjusted odds ratio of spontaneous abortion was 2.69 [95% confidence interval (CI) = 1.33-5.42] for those with genotype Aa or aa in exon 3; but it was 0.45 (95% CI = 0.22-0.94) for those with genotype Aa or aa in exon 4, indicating that the two variants have opposite associations with spontaneous abortion. The findings persisted after adjustment for age, education, parity, smoking, alcohol use, occupation, and pesticide exposure, as well as in subgroup analysis. Moreover, for the variant genotypes Aa or aa in exon 3, the odds ratio was twice as great in those cases with three or more spontaneous abortions than in those with fewer spontaneous abortions.
The crystal structure of recombinant murine liver cytosolic epoxide hydrolase (EC 3.3.2.3) has been determined at 2.8-A resolution. The binding of a nanomolar affinity inhibitor confirms the active site location in the C-terminal domain; this domain is similar to that of haloalkane dehalogenase and shares the alpha/beta hydrolase fold. A structure-based mechanism is proposed that illuminates the unique chemical strategy for the activation of endogenous and man-made epoxide substrates for hydrolysis and detoxification. Surprisingly, a vestigial active site is found in the N-terminal domain similar to that of another enzyme of halocarbon metabolism, haloacid dehalogenase. Although the vestigial active site does not participate in epoxide hydrolysis, the vestigial domain plays a critical structural role by stabilizing the dimer in a distinctive domain-swapped architecture. Given the genetic and structural relationships among these enzymes of xenobiotic metabolism, a structure-based evolutionary sequence is postulated.
Racemic fenvaleric acid [2-(4-chlorophenyl)-3-methyl-butanoic acid], the principal metabolite of fenvalerate, was administrated orally at 0.75, 1.5, and 3.0 mmol/kg body weight/day to Fisher-344 male rats for 7 days. Both pure enantiomers of fenvaleric acid were administered at 1.5 mmol/kg body weight/day; the clofibric acid at the same concentration was used as a positive control. Hepatic enzyme activities were measured. Results obtained clearly show that fenvaleric acid induced numerous hepatic drug metabolism enzymes in F344 rats. The (R) enantiomer of this compound induces a proliferation of peroxisomes, whereas the (S) enantiomer induces CYP2B and mEH activities. Therefore, high exposure to pyrethroid insecticides could interact with the normal metabolism of drugs or xenobiotics.
C-2′,3′); fast atom bombardment MS m/z (relative intensity
- + + Cdo
CdO), 40.2 (C-1), 31.7 (C-6), 30.3 (C-2), 29.2 (C-4), 29.1 (C-5), 26.8 (C-3), 22.5 (C-7), 22.3 (C-1′), 13.9 (C-8), 7.1 (C-2′,3′); fast atom bombardment MS m/z (relative intensity) 425 (2M + H +, 16%), 213 (M + H +, 86%), 58 (cyclopropyl-NH2 + H +, 100%).
0.7 (m, 2 H, cyclopropyl), 0.5 (m, 2 H, cyclopropyl)
- Hz
- Ch
Hz, CH3), 0.7 (m, 2 H, cyclopropyl), 0.5 (m, 2 H, cyclopropyl); 13 C NMR (CDCl3) δ
0) at 30 °C with the RmEH diluted to give a maximal rate of 0 Membrane-bound and soluble-fraction epoxide hydrolases: methodological aspects: Method-ological aspects of drug metabolizing enzymes
- Tris Wixtrom
- R N Hammock
Tris/HCl buffer (pH 9.0) at 30 °C with the RmEH diluted to give a maximal rate of 0.4 References (1) Wixtrom, R. N., and Hammock, B. D. (1985) Membrane-bound and soluble-fraction epoxide hydrolases: methodological aspects. In Biochemical Pharmacology and Toxicology Vol. 1: Method-ological aspects of drug metabolizing enzymes (Zakim, D., and Vessey, D. A., Eds.) pp 1-93, John Wiley & Sons, New York.
Membrane-bound and soluble-fraction epoxide hydrolases: methodological aspects
- R N Wixtrom
- B D Hammock
Wixtrom, R. N., and Hammock, B. D. (1985) Membrane-bound
and soluble-fraction epoxide hydrolases: methodological aspects.
In Biochemical Pharmacology and Toxicology Vol. 1: Methodological aspects of drug metabolizing enzymes (Zakim, D., and
Vessey, D. A., Eds.) pp 1-93, John Wiley & Sons, New York.