Soluble epoxide hydrolase inhibition impairs triggering receptor expressed on myeloid cells-1 in periodontal tissue.

AIMS: Periodontitis is a prevalent inflammatory disorder affecting the oral cavity, driven by dysbiotic oral biofilm and host immune response interactions. While the major clinical focus of periodontitis treatment is currently controlling oral biofilm, understanding the immune response is crucial to prevent disease progression. Soluble epoxide hydrolase (sEH) inhibition has shown promise in preventing alveolar bone resorption. Triggering receptors expressed on myeloid cells (TREMs) play pivotal roles in regulating inflammation and bone homeostasis, and dysregulation of TREM signaling is implicated in periodontitis. Here, we investigated the impact of sEH inhibition on TREM 1 and 2 expression, associated with inflammatory cytokines, and histologically assessed the inflammatory infiltrate in periodontal tissue. METHODS: The experimental periodontitis model was induced by placing a ligature around the upper second molar. For 14 days, animals were treated daily with a sEH inhibitor (TPPU) or vehicle. The alveolar bone loss was examined using a methylene blue stain. Gingival tissues were used to measure the mRNA expression of TREM-1, TREM-2, IKKß, NF-κB, IL-1ß, IL-6, IL-8, and TNF-α by RT-qPCR. Another set of experiments was performed to determine the histological inflammatory scores. RESULTS: In a ligature-induced periodontitis model, sEH inhibition prevented alveolar bone loss and reduced TREM1 expression, albeit with a slight elevation compared to the disease-free group. In contrast, TREM2 expression remained elevated, suggesting sustained immunomodulation favoring resolution. The inhibition of sEH reduced the expression of NF-κB, IL-1ß, and TNF-α, while no differences were found in the expression of IL-6, IL-8, and IKKß. In histological analysis, sEH inhibition reduced the inflammatory leukocyte infiltrate in periodontal tissues close to the ligature. CONCLUSION: These findings underscore the potential of sEH inhibition to modulate periodontal inflammation by regulating TREM-1 alongside decreased IL-1ß and TNF-α expression, highlighting a promising therapeutic approach for periodontitis management.

Upregulated Nuclear Expression of Soluble Epoxide Hydrolase Predicts Poor Outcome in Breast Cancer Patients: Importance of the Digital Pathology Approach.

Breast cancer (BC) is the most common cancer in women, with incidence rates increasing globally in recent years. Therefore, it is important to find new molecules with prognostic and therapeutic value to improve therapeutic response and quality of life. The polyunsaturated fatty acids (PUFAs) metabolic pathway participates in various physiological processes, as well as in the development of malignancies. Although aberrancies in the PUFAs metabolic pathway have been implicated in carcinogenesis, the functional and clinical relevance of this pathway has not been well explored in BC. To evaluate the clinical significance of soluble epoxide hydrolase (EPHX2) expression in Mexican patients with BC using tissue microarrays (TMAs) and digital pathology (DP). Immunohistochemical analyses were performed on 11 TMAs with 267 BC samples to quantify this enzyme. Using DP, EPHX2 protein expression was evaluated solely in tumor areas. The association of EPHX2 with overall survival (OS) was detected through bioinformatic analysis in public databases and confirmed in our cohort via Cox regression analysis. Clear nuclear expression of EPHX2 was identified. Receiver operating characteristics (ROC) curves revealed the optimal cutoff point at 2.847062 × 10-3 pixels, with sensitivity of 69.2% and specificity of 67%. Stratification based on this cutoff value showed elevated EPHX2 expression in multiple clinicopathological features, including older age and nuclear grade, human epidermal growth factor receptor 2 (HER2) and triple negative breast cancer (TNBC) subtypes, and recurrence. Kaplan-Meier curves demonstrated how higher nuclear expression of EPHX2 predicts shorter OS. Consistently, multivariate analysis confirmed EPHX2 as an independent predictor of OS, with a hazard ratio (HR) of 3.483 and a 95% confidence interval of 1.804-6.724 (p < 0.001). Our study demonstrates for the first time that nuclear overexpression of EPHX2 is a predictor of poor prognosis in BC patients. The DP approach was instrumental in identifying this significant association. Our study provides valuable insights into the potential clinical utility of EPHX2 as a prognostic biomarker and therapeutic target in BC.

Simultaneous silencing of juvenile hormone metabolism genes through RNAi interrupts metamorphosis in the cotton boll weevil.

The cotton boll weevil (CBW) (Anthonomus grandis) is one of the major insect pests of cotton in Brazil. Currently, CBW control is mainly achieved by insecticide application, which is costly and insufficient to ensure effective crop protection. RNA interference (RNAi) has been used in gene function analysis and the development of insect control methods. However, some insect species respond poorly to RNAi, limiting the widespread application of this approach. Therefore, nanoparticles have been explored as an option to increase RNAi efficiency in recalcitrant insects. Herein, we investigated the potential of chitosan-tripolyphosphate (CS-TPP) and polyethylenimine (PEI) nanoparticles as a dsRNA carrier system to improve RNAi efficiency in the CBW. Different formulations of the nanoparticles with dsRNAs targeting genes associated with juvenile hormone metabolism, such as juvenile hormone diol kinase (JHDK), juvenile hormone epoxide hydrolase (JHEH), and methyl farnesoate hydrolase (MFE), were tested. The formulations were delivered to CBW larvae through injection (0.05-2 µg), and the expression of the target genes was evaluated using RT-qPCR. PEI nanoparticles increased targeted gene silencing compared with naked dsRNAs (up to 80%), whereas CS-TPP-dsRNA nanoparticles decreased gene silencing (0%-20%) or led to the same level of gene silencing as the naked dsRNAs (up to 50%). We next evaluated the effects of targeting a single gene or simultaneously targeting two genes via the injection of naked dsRNAs or dsRNAs complexed with PEI (500 ng) on CBW survival and phenotypes. Overall, the gene expression analysis showed that the treatments with PEI targeting either a single gene or multiple genes induced greater gene silencing than naked dsRNA (∼60%). In addition, the injection of dsJHEH/JHDK, either naked or complexed with PEI, significantly affected CBW survival (18% for PEI nanoparticles and 47% for naked dsRNA) and metamorphosis. Phenotypic alterations, such as uncompleted pupation or malformed pupae, suggested that JHEH and JHDK are involved in developmental regulation. Moreover, CBW larvae treated with dsJHEH/JHDK + PEI (1,000 ng/g) exhibited significantly lower survival rate (55%) than those that were fed the same combination of naked dsRNAs (30%). Our findings demonstrated that PEI nanoparticles can be used as an effective tool for evaluating the biological role of target genes in the CBW as they increase the RNAi response.

Gene expression changes in Epinephelus marginatus (Teleostei, Serranidae) liver reveals candidate molecular biomarker of iron ore contamination.

Wastes from iron ore mining activities are potentially damaging to adjacent aquatic ecosystems. We aimed to determine biomarkers of environmental exposure to this xenobiotic in the dusky grouper Epinephelus marginatus by differential gene expression analysis. For this, fish were exposed to iron ore (15.2 mg/L) and gene expression in liver was assessed by RNA-Seq and compared to the control group. A total of 124 differentially expressed genes were identified, from which 52 were upregulated and 72 were downregulated in response to iron ore. From these, ferritin (medium subunit), cytochrome b reductase and epoxide hydrolase genes were selected for validation by RT-qPCR that confirmed the upregulation of epoxide hydrolase in fish exposed to iron ore.

Polymorphisms in xenobiotic metabolism-related genes in patients with hepatocellular carcinoma: a case-control study.

This study was performed to investigate the relationship between polymorphisms in microsomal epoxide hydrolase (mEH; Tyr113His and His139Arg substitution) and glutathione S-transferase (GST; GSTM1 deletion, GSTT1 deletion, and GSTP1.Ala114Val substitution) and their correlation with clinico-histopathological features in hepatocellular carcinoma (HCC).We evaluated environmental risk factors and genetic alterations in 556 individuals (86 cases and 470 controls). PCR multiplex for GSTM1 and GSTT1, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) for GSTP1, and real-time PCR for mEH were performed. Statistical analyses were performed using multiple logistic regression tests.Age over 48 years (p < 0.001) and alcohol consumption (p = 0.021) were the predictors of increased risk of developing HCC. GSTP1.Ala114Val for all regression models (p < 0.05), except the recessive model, and the GSTT1 null genotype (odds ratio [OR] = 0.43, 95% confidence interval [CI] = 0.21-0.87, p = 0.019) were predictors of an increased risk of developing HCC. Polymorphic GSTT1, GSTM1, GSTP1.Ala114Val, and mEH.His139Arg and wild-type mEH.Tyr113His (OR = 5.04; 95% CI = 1.59-16.04; p = 0.006) were associated with HCC.Age over 48 years, alcohol consumption, and the presence of polymorphic variants of GSTP1 and GSTT1 were associated with the risk of developing HCC.

Peripheral soluble epoxide hydrolase inhibition reduces hypernociception and inflammation in albumin-induced arthritis in temporomandibular joint of rats.

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial tissue, joint dysfunction, and damage. Epoxyeicosatrienoic acids (EETs) are endogenous anti-inflammatory compounds, which are quickly converted by the soluble epoxide hydrolase (sEH) enzyme into a less active form with decreased biological effects. The inhibition of the sEH enzyme has been used as a strategy to lower nociception and inflammation. The goal of this study was to investigate whether the peripheral treatment with the sEH enzyme inhibitor 1- trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) could prevent the hypernociception and inflammation in the albumin-induced arthritis model in rats' temporomandibular joint (TMJ). After the induction of experimental arthritis, animals were assessed for nociceptive behavior test, leukocyte infiltration counts and histologic analysis, ELISA to quantify several cytokines and Western blotting. The peripheral pretreatment with TPPU inhibited the arthritis-induced TMJ hypernociception and leukocyte migration. Moreover, the local concentrations of proinflammatory cytokines were diminished by TPPU, while the anti-inflammatory cytokine interleukin-10 was up-regulated in the TMJ tissue. Finally, TPPU significantly decreased protein expression of iNOS, while did not alter the expression of MRC1. This study provides evidence that the peripheral administration of TPPU reduces hypernociception and inflammation in TMJ experimental arthritis.

Soluble epoxide hydrolase inhibitor, TPPU, increases regulatory T cells pathway in an arthritis model.

Epoxyeicosatrienoic acids (EET) and related epoxy fatty acids (EpFA) are endogenous anti-inflammatory compounds, which are converted by the soluble epoxide hydrolase (sEH) to dihydroxylethersatrienoic acids (DHETs) with lessened biological effects. Inhibition of sEH is used as a strategy to increase EET levels leading to lower inflammation. Rheumatoid arthritis is a chronic autoimmune disease that leads to destruction of joint tissues. This pathogenesis involves a complex interplay between the immune system, and environmental factors. Here, we investigate the effects of inhibiting sEH with 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) on a collagen-induced arthritis model. The treatment with TPPU ameliorates hyperalgesia, edema, and decreases the expression of important pro-inflammatory cytokines of Th1 and Th17 profiles, while increasing Treg cells. Considering the challenges to control RA, this study provides robust data supporting that inhibition of the sEH is a promising target to treat arthritis.

The molecular structure of an epoxide hydrolase from Trichoderma reesei in complex with urea or amide-based inhibitors.

Epoxide hydrolases (EHs) are enzymes involved in the metabolism of endogenous and exogenous epoxides, and the development of EH inhibitors has important applications in the medicine. In humans, EH inhibitors are being tested in the treatment of cardiovascular diseases and show potent anti-inflammatory effects. EH inhibitors are also considerate promising molecules against infectious diseases. EHs are functionally very well studied, but only a few members have its three-dimensional structures characterized. Recently, a new EH from the filamentous fungi Trichoderma reseei (TrEH) was reported, and a series of urea or amide-based inhibitors were identified. In this study, we describe the crystallographic structures of TrEH in complex with five different urea or amide-based inhibitors with resolutions ranging from 2.6 to 1.7 Å. The analysis of these structures reveals the molecular basis of the inhibition of these compounds. We could also observe that these inhibitors occupy the whole extension of the active site groove and only a few conformational changes are involved. Understanding the structural basis EH interactions with different inhibitors might substantially contribute for the study of fungal metabolism and in the development of novel and more efficient antifungal drugs against pathogenic Trichoderma species.

Substrate and inhibitor selectivity, and biological activity of an epoxide hydrolase from Trichoderma reesei.

Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EH are involved in the metabolism of endogenous and exogenous epoxides, and thus have application in pharmacology and biotechnology. In this work, we describe the substrates and inhibitors selectivity of an epoxide hydrolase recently cloned from the filamentous fungus Trichoderma reesei QM9414 (TrEH). We also studied the TrEH urea-based inhibitors effects in the fungal growth. TrEH showed high activity on radioative and fluorescent surrogate and natural substrates, especially epoxides from docosahexaenoic acid. Using a fluorescent surrogate substrate, potent inhibitors of TrEH were identified. Interestingly, one of the best compounds inhibit up to 60% of T. reesei growth, indicating an endogenous role for TrEH. These data make TrEH very attractive for future studies about fungal metabolism of fatty acids and possible development of novel drugs for human diseases.

Soluble Epoxide Hydrolase and Brain Cholesterol Metabolism.

The bifunctional enzyme soluble epoxide hydrolase (sEH) is found in all regions of the brain. It has two different catalytic activities, each assigned to one of its terminal domains: the C-terminal domain presents hydrolase activity, whereas the N-terminal domain exhibits phosphatase activity. The enzyme's C-terminal domain has been linked to cardiovascular protective and anti-inflammatory effects. Cholesterol-related disorders have been associated with sEH, which plays an important role in the metabolism of cholesterol precursors. The role of sEH's phosphatase activity has been so far poorly investigated in the context of the central nervous system physiology. Given that brain cholesterol disturbances play a role in the onset of Alzheimer's disease (AD) as well as of other neurodegenerative diseases, understanding the functions of this enzyme could provide pivotal information on the pathophysiology of these conditions. Moreover, the sEH phosphatase domain could represent an underexplored target for drug design and therapeutic strategies to improve symptoms related to neurodegenerative diseases. This review discusses the function of sEH in mammals and its protein structure and catalytic activities. Particular attention was given to the distribution and expression of sEH in the human brain, deepening into the enzyme's phosphatase activity and its participation in brain cholesterol synthesis. Finally, this review focused on the metabolism of cholesterol and its association with AD.

Soluble epoxide hydrolase inhibitor promotes immunomodulation to inhibit bone resorption.

BACKGROUND AND OBJECTIVE: Soluble epoxide hydrolase (sEH) is an enzyme in the arachidonate cascade which converts epoxy fatty acids (EpFAs), such as epoxyeicosatrienoic acids (EETs) produced by cytochrome P450 enzymes, to dihydroxy-eicosatrienoic acids. In the last 20 years with the development of inhibitors to sEH it has been possible to increase the levels of EETs and other EpFAs in in vivo models. Recently, studies have shown that EETs play a key role in blocking inflammation in a bone resorption process, but the mechanism is not clear. In the current study we used the sEH inhibitor (1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea [TPPU]) to investigate the immunomodulatory effects in a mouse periodontitis model. MATERIAL AND METHODS: Mice were infected on days 0, 2, and 4 with Aggregatibacter actinomycetemcomitans and divided into groups (n = 6) that were treated orally, daily for 15 days, with 1 mg/kg of TPPU. Then, the mice were killed and their jaws were analyzed for bone resorption using morphometry. Immunoinflammatory markers in the gingival tissue were analyzed by microarray PCR or western blotting. RESULTS: Infected mice treated with TPPU showed lower bone resorption than infected mice without treatment. Interestingly, infected mice showed increased expression of sEH; however, mice treated with TPPU had a reduction in expression of sEH. Besides, several proinflammatory cytokines and molecular markers were downregulated in the gingival tissue in the group treated with 1 mg/kg of TPPU. CONCLUSION: The sEH inhibitor, TPPU, showed immunomodulatory effects, decreasing bone resorption and inflammatory responses in a bone resorption mouse model.

Role of CYP2C9, CYP2C19 and EPHX Polymorphism in the Pharmacokinetic of Phenytoin: A Study on Uruguayan Caucasian Subjects.

Phenytoin (PHT) oxidative route leads to its main metabolite p-hydroxyphenytoin (p-HPPH), by means of CYP2C9 and CYP2C19. Formation of p-HPPH proceeds via a reactive arene-oxide intermediate. This intermediate can also be converted into PHT dihydrodiol by microsomal epoxide hydrolase (EPHX). The three enzymes are polymorphically expressed and the genetic variants are responsible for changes in the enzyme activity. In order to evaluate the effect that these polymorphisms have on PHT metabolism, PHT and p-HPPH plasma concentrations were measured and the genotype for the three enzymes was assessed in 50 Uruguayan epileptic patients. 30% of the patients were intermediate and 2% were poor metabolizers for CYP2C9, while 20% were intermediate metabolizers for CYP2C19. 44%, 10%, and 46% of subjects had intermediate, increased and decreased activities of EPHX respectively. CYP2C9 was confirmed to be the main responsible enzyme for PHT biotransformation. CYP2C19 seemed to be preponderant in p-HPPH oxidative metabolism. Apart from being responsible for the production of the dihydrodiol metabolite, EPHX also seemed to contribute to pHPPH formation when its activity is low. PHT might be recovered with a decreased activity of EPHX regardless the activity of CYP2C9.

Structure of a soluble epoxide hydrolase identified in Trichoderma reesei.

Epoxide hydrolases (EHs) are enzymes that have high biotechnological interest for the fine and transformation industry. Several of these enzymes have enantioselectivity, which allows their application in the separation of enantiomeric mixtures of epoxide substrates. Although two different families of EHs have been described, those that have the α/ß-hidrolase fold are the most explored for biotechnological purpose. These enzymes are functionally very well studied, but only few members have three-dimensional structures characterised. Recently, a new EH from the filamentous fungi Trichoderma reseei (TrEH) has been discovered and functionally studied. This enzyme does not have high homology to any other EH structure and have an enatiopreference for (S)-(-) isomers. Herein we described the crystallographic structure of TrEH at 1.7Å resolution, which reveals features of its tertiary structure and active site. TrEH has a similar fold to the other soluble epoxide hydrolases and has the two characteristic hydrolase and cap domains. The enzyme is predominantly monomeric in solution and has also been crystallised as a monomer in the asymmetric unit. Although the catalytic residues are conserved, several other residues of the catalytic groove are not, and might be involved in the specificity for substrates and in the enantioselectivy of this enzyme. In addition, the determination of the crystallographic structure of TrEH might contribute to the rational site direct mutagenesis to generate an even more stable enzyme with higher efficiency to be used in biotechnological purposes.

Structural insights into human microsomal epoxide hydrolase by combined homology modeling, molecular dynamics simulations, and molecular docking calculations.

A new homology model of human microsomal epoxide hydrolase was derived based on multiple templates. The model obtained was fully evaluated, including MD simulations and ensemble-based docking, showing that the quality of the structure is better than that of only previously known model. Particularly, a catalytic triad was clearly identified, in agreement with the experimental information available. Analysis of intermediates in the enzymatic mechanism led to the identification of key residues for substrate binding, stereoselectivity, and intermediate stabilization during the reaction. In particular, we have confirmed the role of the oxyanion hole and the conserved motif (HGXP) in epoxide hydrolases, in excellent agreement with known experimental and computational data on similar systems. The model obtained is the first one that fully agrees with all the experimental observations on the system. Proteins 2017; 85:720-730. © 2016 Wiley Periodicals, Inc.

Data set of optimal parameters for colorimetric red assay of epoxide hydrolase activity.

The data presented in this article are related to the research article entitled "Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization" [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay.

Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization.

Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have biotechnological potential in chiral chemistry. We report the cloning, purification, enzymatic activity, and conformational analysis of the TrEH gene from Trichoderma reesei strain QM9414 using circular dichroism spectroscopy. The EH gene has an open reading frame encoding a protein of 343 amino acid residues, resulting in a molecular mass of 38.2kDa. The enzyme presents an optimum pH of 7.2, and it is highly active at temperatures ranging from 23 to 50°C and thermally inactivated at 70°C (t1/2=7.4min). The Michaelis constants (Km) were 4.6mM for racemic substrate, 21.7mM for (R)-(+)-styrene oxide and 3.0mM for (S)-(-)-styrene oxide. The kcat/Km analysis indicated that TrEH is enantioselective and preferentially hydrolyzes (S)-(-)-styrene oxide. The conformational stability studies suggested that, despite the extreme conditions (high temperatures and extremely acid and basic pHs), TrEH is able to maintain a considerable part of its regular structures, including the preservation of the native cores in some cases. The recombinant protein showed enantioselectivity that was distinct from other fungus EHs, making this protein a potential biotechnological tool.

The GSTM1null (deletion) and MGMT84 rs12917 (Phe/Phe) haplotype are associated with bulky DNA adduct levels in human leukocytes.

Tobacco smoke and air pollutants contain carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and tobacco specific nitrosamines (TSNA), that are substrates of metabolizing enzymes generating reactive metabolites that can bind to DNA. Variation in the activity of these enzymes may modify the extent to which these metabolites can interact with DNA. We compared the levels of bulky DNA adducts in blood leukocytes from 93 volunteers living in Mexico City with the presence of 13 single nucleotide polymorphisms (SNPs) in genes related to PAH and TSNA metabolism (AhR rs2044853, CYP1A1 rs1048943, CYP1A1 rs1048943, CYP1A1 rs1799814, EPHX1 rs1051740, EPHX1 rs2234922, GSTM1 null, GSTT1 null and GSTP1 rs947894), DNA repair (XRCC1 rs25487, ERCC2 rs13181 and MGMT rs12917) and cell cycle (TP53 rs1042522). (32)P-postlabeling analysis was used to quantify bulky DNA adduct formation. Genotyping was performed using PCR-RFLP. The mean levels of bulky DNA adducts were 8.51±3.66 adducts/10(8) nucleotides (nt) in smokers and 8.38±3.59 adducts/10(8) nt in non-smokers, being the difference not statistically significant. Without taking into account the smoking status, GSTM1 null individuals had a marginally significant lower adduct levels compared with GSTM1 volunteers (p=0.0433) and individuals heterozygous for MGMT Leu/Phe had a higher level of bulky adducts than those who were homozygous wild type (p=0.0170). A multiple regression analysis model showed a significant association between the GSTM1 (deletion) and MGMT rs12917 (Phe/Phe) haplotype and the formation of DNA adducts in smokers (R(2)=0.2401, p=0.0215). The presence of these variants conferred a greater risk for higher adduct levels in this Mexican population.