Cytochrome P450 2J2 Research Articles

Potential of hydroxyebastine and terfenadine alcohol to inhibit the human cytochrome P450 2J2 isoform

Although selective inhibitors of cytochrome P450 enzymes can be used to determine relative contributions of the enzymes to xenobiotic metabolism, characterization of CYP2J2 in drug metabolism is more challenging due to lack of selective, well-characterized inhibitors. Thus, selectivity of hydroxyebastine, which has high affinity for recombinant CYP2J2, was studied. The IC50 value of hydroxyebastine in CYP2J2-mediated astemizole O-demethylation activity was lower than that of its structural analog, terfenadine alcohol. Terfenadine alcohol inhibited several other P450 activities, such as CYP2D6, more potently than CYP2J2, and is thus not suitable as a CYP2J2-selective inhibitor. Inhibitory potential values of hydroxyebastine in CYP2J2-catalyzed astemizole O-demethylation, tolbutamide hydroxylation (CYP2C9), S-mephenytoin hydroxylation (CYP2C19), and dextromethorphan O-demethylation (CYP2D6) were 0.45, 2.74, 10.22, and 3.83 μM, respectively. The inhibitory potential of other P450 enzymes, such as CYP1A2, CYP2B6, CYP2E1, and CYP3A, was negligible. Although hydroxyebastine was a relatively potent inhibitor of CYP2J2, it provided a selectivity of only > 6-fold (CYP2J2 vs. other P450s). However, hydroxyebastine can serve as a relatively selective inhibitor of CYP2J2 and can be used to characterize the contribution of CYP2J2 to xenobiotic metabolism due to the lack of a more specific inhibitor.

Association between polymorphisms of CYP2J2 and EPHX2 genes and risk of coronary artery disease

Common polymorphisms within cytochrome P450 2J2 (CYP2J2) and epoxide hydrolase 2 (EPHX2), which are involved in the generation or hydrolysis of epoxyeicosatrienoic acids, may determine susceptibility to the development of cardiovascular disease. To derive a more precise estimation of their relationship, we undertook a case-control study as well as a meta-analysis to assess possible associations of coronary artery disease (CAD) risk with CYP2J2 and EPHX2 genetic variations. Associations among four single nucleotide polymorphisms in CYP2J2 and five in EPHX2 with CAD were examined in a total of 1344 cases and 1267 ethnically and geographically matched controls. To further confirm the effect of two functional variants (G-50T and R287Q) in the development of CAD, we conducted a meta-analysis including seven studies on G-50T polymorphism and six studies on R287Q polymorphism before June 2010. No significant association between common polymorphisms within these two genes and CAD was observed in our sample, either using methods of single-locus analysis or haplotype-based analysis. In addition, no association was detected in our meta-analysis between these two functional variants and the risk of developing CAD. This case-control study as well as meta-analysis suggested no association between CYP2J2 G-50T and EPHX2 R287Q and the risk of developing CAD.

The shunting of arachidonic acid metabolism to 5-lipoxygenase and cytochrome p450 epoxygenase antagonizes the anti-cancer effect of cyclooxygenase-2 inhibition in head and neck cancer cells

It has recently been found that 5-lipoxygenase (5-LO) and cytochrome P450-2J2 (CYP2J2), molecules capable of arachidonic acid (AA) metabolism, might promote cancer cell viability through several mechanisms similar to those of cyclooxygenase-2 (COX-2). We found that not only COX-2 expression, but also the expression of 5-LO and CYP2J2 is up-regulated in head and neck squamous cell carcinoma (HNSCC) cell lines. From these observations, we hypothesized that AA metabolism by 5-LO and/or CYP2J2 may lower the efficacy of anti-cancer effect by COX-2 inhibition. Although COX-2 was highly expressed in all cell lines tested, COX-2-specific inhibition showed little growth-inhibitory effect in some cell lines. Inhibition of COX-2 resulted in increased production of LTB(4) and 14-15-DHET/EET, metabolites of 5-LO and CYP2J2, respectively. Combined knock-down of COX-2 and 5-LO or CYP2J2 by siRNA results in a decrease in cell proliferation and VEGF production. Furthermore, these results are dependent on 5-LO and CYP2J2 expression in cells. Therefore, combined inhibition of COX-2 and 5-LO or CYP2J2 may be one way to overcome low efficacy of single inhibition of COX-2 in cancer cells. In addition, combined therapies should be chosen based on the expression of members of other AA metabolism pathways.

Cytochrome P450 2J2 Is Highly Expressed in Hematologic Malignant Diseases and Promotes Tumor Cell Growth

Cytochrome P450 2J2 (CYP2J2) epoxygenase converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs) that exert multiple biological effects in the cardiovascular system and in various human solid cancers. However, it is unknown whether this enzyme is expressed or plays any role in malignant hematological diseases. In this study, we found strong and highly selective CYP2J2 expression in five human-derived malignant hematological cell lines and in leukemia cells from peripheral blood and bone marrow in 36 of 42 patients (86%) with malignant hematologic diseases. Furthermore, increased levels of EETs were detected in urine and blood samples from these patients. Addition of exogenous EET or CYP2J2 overexpression in cultured human-derived malignant hematologic cell lines markedly accelerated proliferation and attenuated apoptosis. Addition of the selective CYP2J2 inhibitor compound 26 (C26; 1-[4-(vinyl) phenyl]-4-[4-(diphenyl-hydroxymethyl)-piperidinyl]-butanone hydrochloride) inhibited cell proliferation and increased apoptosis, an effect that was significantly reversed by EET. CYP2J2 overexpression and exogenous EET activated AMP-activated protein kinase, c-Jun NH(2)-terminal kinase, and phosphatidylinositol 3-kinase/Akt signaling pathways, and increased epidermal growth factor receptor phosphorylation levels. CYP2J2 overexpression also enhanced malignant xenograft growth, which was efficiently inhibited by oral administration of C26 in Tie2-CYP2J2 transgenic mice and in severe combined immunodeficiency (SCID) xenograft mice. Together, these results suggest that CYP2J2 plays a key role in the pathogenesis of human hematologic malignant diseases. Selective inhibition of CYP2J2 may be a promising therapeutic strategy for these conditions.

Inhibition of carcinoma cell motility by epoxyeicosatrienoic acid (EET) antagonists

Cytochrome P450 (CYP) epoxygenases, CYP2C8, 2C9 and 2J2 mRNA and proteins, were expressed in prostate carcinoma (PC-3, DU-145 and LNCaP) cells. 11,12-Epoxyeicosatrienoic acid (11,12-EET) was the major arachidonic acid metabolite in these cells. Blocking EET synthesis by a selective CYP epoxygenase inhibitor (N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide [MS-PPOH]) inhibited tonic (basal) invasion and migration (motility) while exogenously added EET induced cell motility in a concentration-dependent manner. An epidermal growth factor receptor (EGFR) kinase inhibitor (AG494) or a PI3 kinase inhibitor (LY294002) inhibited cell migration and reduced 11,12-EET-induced cell migration. Importantly, synthetic EET antagonists (14,15-epoxyeicosa-5(Z)-enoic acid [14,15-EEZE], 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxy-propoxy)-ethoxy]-ethyl ester [14,15-EEZE-PEG] and 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide [14,15-EEZE-mSI]) inhibited EET-induced cell invasion and migration. 11,12-EET induced cell stretching and myosin-actin microfilament formation as well as increased phosphorylation of EGFR and Akt (Ser473), while 14,15-EEZE inhibited these effects. These results suggest that EET induce and EET antagonists inhibit cell motility, possibly by putative EET receptor-mediated EGFR and PI3K/Akt pathways, and suggest that EET antagonists are potential therapeutic agents for prostate cancer.

Impaired transactivation of the human CYP2J2 arachidonic acid epoxygenase gene in HepG2 cells subjected to nitrative stress

Human cytochrome P450 2J2 (CYP2J2) generates epoxyfatty acids that modulate cellular apoptosis and proliferation. CYP2J2 regulation has not been intensively studied but induction of the activator protein-1 (AP-1) subunit c-fos mediates CYP2J2 down-regulation in hypoxia, a component of ischaemic injury. Decreased CYP2J2 expression may contribute to tissue injury. HepG2 cells were treated with sodium nitroprusside (SNP) to induce nitrative stress, which has been associated with inflammation and infection in liver and other tissues. CYP2J2 protein and mRNA expression were evaluated by immunoblotting and real-time PCR respectively. The role of mitogen-activated protein (MAP) kinases in CYP2J2 dysregulation was assessed using specific inhibitors and dominant negative MAP kinase expression plasmids. CYP2J2-luciferase reporter constructs and electromobility shift assays (EMSAs) were used to identify SNP-regulated regions in the CYP2J2 gene. Cytochrome P450 2J2 was down-regulated by SNP while the AP-1 proteins c-jun and c-fos were up-regulated; inhibition of p38 and ERK MAP kinases normalized their expression. The gene elements at -105/-95 and -56/-63 were required for the down-regulation of CYP2J2 induced by nitrative stress. p38 and ERK MAP kinases transduce stress stimuli that down-regulate CYP2J2. Targeting these kinases may prevent the loss of CYP2J2 and epoxy-fatty acids that protect cells against deleterious stresses.

Up-Regulation of Human CYP2J2 in HepG2 Cells by Butylated Hydroxyanisole Is Mediated by c-Jun and Nrf2

Cytochrome P450 2J2 oxidizes arachidonic acid to a series of epoxyeicosatrienoic acid (EET) isomers in human tissues. EETs regulate numerous homeostatic processes, including cytoprotective and proliferative responses against injurious stresses. There is little information currently available on the factors that regulate CYP2J2, but strategies to activate expression could use the beneficial effects of EETs in cells. The basic leucine zipper (bZIP) transcription factor c-Jun has been shown previously to maintain CYP2J2 expression in human HepG2 cells; c-Jun forms transcriptionally active dimers with the antioxidant-inducible bZIP factor Nrf2. In the present study, we tested the hypothesis that CYP2J2 expression may be activated in cells by c-Jun/Nrf2 heterodimers. Treatment of HepG2 cells with butylated hydroxyanisole elicited concentration- and time-dependent activation of CYP2J2 expression, as well as the bZIP factors Nrf2 and c-Jun; chromatin immunoprecipitation assays revealed a pronounced increase in binding of these bZIP factors to the CYP2J2 5'-flank. Transient transfection analysis using deletion constructs and gel-shift assays were consistent with a role for the -105/-88 region of CYP2J2 in c-Jun/Nrf2 responsiveness. Using a series of mutant expression plasmids, we identified c-Jun as the critical partner in CYP2J2 transactivation. Coimmunoprecipitation experiments confirmed the importance of the leucine zipper region of Nrf2 in the enhancement of c-Jun-dependent transactivation of CYP2J2. Agents that activate CYP2J2 expression may offer a new approach to using the beneficial effects of EETs in cells.

Genetic Variation in the G-50T Polymorphism of the Cytochrome P450 Epoxygenase CYP2J2 Gene and the Risk of Younger Onset Type 2 Diabetes among Chinese Population: Potential Interaction with Body Mass Index and Family History

Cytochrome P450 (CYP) 2J2 is a regulatory enzyme in the biosynthesis of biologically active CIS-epoxyeicosatrienoic acids (EETs). EETs have been suggested to modulate PPAR-gamma and PPAR-alpha transcription activity and play a role in stimulus-secretion coupling in pancreatic beta cells. Genetic abnormalities in the expression of CYP2J enzymes may play a role in the pathogenesis of type 2 diabetes mellitus (T2DM). Our objective was to investigate CYP2J2 G-50T polymorphism (rs890293) in association with insulin resistance markers and T2DM in a Chinese population. A total of 1 747 Chinese T2DM patients and 994 non-diabetic subjects were studied. The CYP2J2 G-50T polymorphism was determined by a restriction fragment-length polymorphism polymerase chain reaction. Neither the CYP2J2 genotype distribution nor allele frequency differed between the control subjects and the T2DM patients. However, among diabetics, subjects with a younger age at diagnosis (AAD; <40 years) had significantly higher T variant frequency than those with an AAD>/=40 years. When diabetic patients were stratified by their AAD in 10-year intervals, the trend was significantly linear among age grades. A significant interaction between the CYP2J2 T variant and younger onset diabetic subjects with positive family diabetes history, and BMI>/=27 kg/m (2) were observed to have the highest risk of diabetes and younger onset diabetics with the T variant had higher homeostasis model assessment estimate of insulin resistance (HOMA-IR) and HOMA-beta values than their GG genotype counterparts. Plasma concentrations of stable EET metabolites were significantly lower in individuals with the G-50T SNP in younger onset diabetics. These data suggest that age of onset, family history, and obesity may modify the association between the CYP2J2 G-50T polymorphism and T2DM risk. CYP2J2 G-50T polymorphism may contribute to the pathogenesis of T2DM, partially by effects on insulin resistance, in patients with younger onset T2DM.

Identification of 13-Hydroxy-14,15-epoxyeicosatrienoic Acid as an Acid-stable Endothelium-derived Hyperpolarizing Factor in Rabbit Arteries

Arachidonic acid (AA) is metabolized by endothelial 15-lipoxygenase (15-LO) to several vasodilatory eicosanoids such as 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) and its proposed unstable precursor 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). In the present study, the acid-stable 13-hydroxy-trans-14,15-epoxy-eicosatrienoic acid (13-H-14,15-EETA) was identified and its vascular activities characterized. Rabbit aorta, mesenteric arteries, and the combination of 15-LO and cytochrome P450 2J2 converted AA to two distinct HEETA metabolites. The HEETA metabolites were resistant to acidic hydrolysis but were hydrolyzed by recombinant sEH to a more polar metabolite identified by mass spectrometry as 13,14,15-THETA. Mass spectrometric analyses and HPLC comigration identified the HEETAs as threo- and erythro-diastereomers of 13-H-trans-14,15-EETA. Erythro- and threo-diastereomers of 13-H-trans-14,15-EETA relaxed endothelium-denuded rabbit small mesenteric arteries with maximum relaxations of 22.6 +/- 6.0% and 8.6 +/- 4.3%, respectively. Apamin (10(-7) m) inhibited the relaxations to the erythro-isomer (maximum relaxation = 1.2 +/- 5.6%) and increasing [K(+)](o) from 4.6 to 30 mm blocked relaxations to both isomers. In cell-attached patches of mesenteric arterial smooth muscle cells (SMCs), erythro-13-H-trans-14,15-EETA (1-3 x 10(-6) m) increased mean open time of small conductance K(+) channels (13-14 pS) from 0.0007 +/- 0.0007 to 0.0053 +/- 0.0042. This activation was inhibited by apamin. The erythro, but not the threo, isomer blocked angiotensin II-stimulated aortic SMC migration. These studies demonstrate that 13-H-14,15-EETAs induces vascular relaxation via K(+) channel activation to cause SMC hyperpolarization. Thus, 13-H-14,15-EETA represents a new endothelial factor.

Cytochrome P450 2J2 gene transgene is protective against experimental cerebral ischemia in mice

Cytochrome P450 2J2 gene transgene is protective against experimental cerebral ischemia in mice

Eicosapentaenoic acid increases cytochrome P-450 2J2 gene expression and epoxyeicosatrienoic acid production via peroxisome proliferator-activated receptor γ in endothelial cells

ω-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on cardiovascular diseases. Cytochrome P-450 (CYP) 2J2 that is expressed in endothelial cells metabolizes arachidonic acids to biologically active epoxyeicosatrienoic acids (EETs) that possess anti-inflammatory and anti-thrombotic effects. We studied the effects of EPA and DHA on the expression of CYP 2J2 mRNA by reverse transcription-polymerase chain reaction in cultured human umbilical vein endothelial cells and found that EPA, but not DHA, increased the expression of CYP 2J2 mRNA in a dose-dependent and a time-dependent manner. EPA-induced CYP 2J2 expression was significantly inhibited by pretreatment with a peroxisome proliferator-activated receptor (PPAR) γ antagonist, GW9662. EPA, but not DHA, caused a significant increase in cellular levels of 11,12-dihydroxyeicosatrienoic acid that is a stable metabolite of 11,12-EET, which was blocked by pretreatment with GW9662. These data demonstrate that EPA increases CYP 2J2 mRNA expression and 11,12-EET production via PPARγ in endothelial cells and indicate a novel protective role of EPA and PPARγ against vascular inflammation.

CYP4A11 polymorphism correlates with coronary endothelial dysfunction in patients with coronary artery disease—The ENCORE Trials

Background Cytochrome P450 (CYP) is expressed in the human endothelium and metabolizes arachidonic acid into vasoactive epoxyeicosatrienoic and 20-hydroxyeicosatetraenoic acids. CYP enzymes have been linked to hypertension and generation of reactive oxygen species. Thus, we investigated the impact of several CYP polymorphisms on coronary endothelial function in patients with coronary artery disease (CAD). Methods and results We determined CYP4A11 F434S, CYP2C9 I359L, CYP2C9 G144C and CYP 2J2 promotor -50G>T polymorphisms in 734 patients with CAD undergoing percutaneous coronary intervention. Increasing concentrations of acetylcholine were infused in a coronary segment without angiographically significant CAD and the coronary artery vasomotor response was measured by quantitative angiography. Patients with substitution of phenylalanine 434 by serine (434SS, n = 15, 2.04%) in CYP4A11 F434 demonstrated significantly augmented endothelium-dependent vasoconstriction ( p = 0.044 after Bonferroni correction) compared to patients with the 434FS ( n = 193, 26.29%) and 434FF genotype ( n = 526, 71.66%) before and after adjustment for blood pressure and HDL-cholesterol. In addition, patients with the 434SS genotype had higher systolic blood pressure levels ( p = 0.039) compared to the two other groups. The CYP 2C9 and CYP 2J2 polymorphisms did not show any correlation with coronary vasoconstriction, hypertension, diabetes mellitus, blood pressure or cholesterol. Conclusion In patients with established and stable coronary artery disease the 434SS variant of CYP4A11 F434 is associated with pronounced coronary vasoconstriction.

Overexpression of CYP2J2 provides protection against doxorubicin-induced cardiotoxicity

Human cytochrome P-450 (CYP)2J2 is abundant in heart and active in biosynthesis of epoxyeicosatrienoic acids (EETs). Recently, we demonstrated that these eicosanoid products protect myocardium from ischemia-reperfusion injury. The present study utilized transgenic (Tr) mice with cardiomyocyte-specific overexpression of human CYP2J2 to investigate protection toward toxicity resulting from acute (0, 5, or 15 mg/kg daily for 3 days, followed by 24-h recovery) or chronic (0, 1.5, or 3.0 mg/kg biweekly for 5 wk, followed by 2-wk recovery) doxorubicin (Dox) administration. Acute treatment resulted in marked elevations of serum lactate dehydrogenase and creatine kinase levels that were significantly greater in wild-type (WT) than CYP2J2 Tr mice. Acute treatment also resulted in less activation of stress response enzymes in CYP2J2 Tr mice (catalase 750% vs. 300% of baseline, caspase-3 235% vs. 165% of baseline in WT vs. CYP2J2 Tr mice). Moreover, CYP2J2 Tr hearts exhibited less Dox-induced cardiomyocytes apoptosis (measured by TUNEL) compared with WT hearts. After chronic treatment, comparable decreases in body weight were observed in WT and CYP2J2 Tr mice. However, cardiac function, assessed by measurement of fractional shortening with M-mode transthoracic echocardiography, was significantly higher in CYP2J2 Tr than WT hearts after chronic Dox treatment (WT 37 +/- 2%, CYP2J2 Tr 47 +/- 1%). WT mice also had larger increases in beta-myosin heavy chain and cardiac ankryin repeat protein compared with CYP2J2 Tr mice. CYP2J2 Tr hearts had a significantly higher rate of Dox metabolism than WT hearts (2.2 +/- 0.25 vs. 1.6 +/- 0.50 ng.min(-1).100 microg protein(-1)). In vitro data from H9c2 cells demonstrated that EETs attenuated Dox-induced mitochondrial damage. Together, these data suggest that cardiac-specific overexpression of CYP2J2 limited Dox-induced toxicity.

Role of cytochrome P450 2C8 and 2J2 genotypes in calcineurin inhibitor-induced chronic kidney disease

The calcineurin inhibitors (CNIs) cyclosporine A (CsA) and tacrolimus (Tac) help prevent allograft rejection but are associated with nephrotoxicity. Cytochrome P450 2C8 (CYP2C8) and CYP2J2 are polymorphic enzymes expressed in the kidney that metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids, promoting kidney homeostasis. This study examined the association between CNI-induced nephrotoxicity in liver transplant patients and CYP2C8 and CYP2J2 polymorphisms. Liver transplantation patients receiving CNIs for at least 3 years were genotyped for CYP2C8*3, CYP2C8*4, CYP2C8 Haplotypes B and C, and CYP2J2*7 and evaluated for nephrotoxicity (serum creatinine > or = 1.6 mg/dl) 3-year post-transplantation. CYP2C8 proteins were also engineered in E. coli and their activity towards AA and inhibition by CNIs was investigated in vitro. The risk of kidney disease post-transplantation was positively associated with CYP2C8*3 genotype. Odds ratios for all participants carrying at least one CYP2C8*3 allele were significant [odds ratio=2.38 (1.19-4.78)]. Stratification by CNI indicated a significant association between CYP2C8*3 and nephrotoxicity among patients receiving Tac but not CsA. The risk of renal dysfunction was not significantly influenced by CYP2C8*4, CYP2J2*7, or CYP2C8 haplotype B genotypes although inheritance of haplotype C seems to be protective. In vitro, the gene products of CYP2C8*3 and CYP2C8*4 were deficient in AA epoxidation, retaining 26 and 18% of wild-type activity, respectively. Circulating plasma concentrations of CsA and Tac inhibited CYP2C8 wild-type in vitro epoxidation of AA by 17 and 35%, respectively. Inheritance of CYP2C8*3 is associated with a higher risk of developing renal toxicity in patients treated chronically with CNIs, and especially Tac, possibly by reducing formation of kidney protecting vasodilatory epoxyeicosatrienoic acids.

Cytochrome P450 2J2*7 polymorphisms in Japanese, Mongolians and Ovambos.

Human cytochrome P450 2J2 (CYP2J2) is abundant in cardiovascular tissue and active in the metabolism of arachidonic acid to eicosanoids that have potent vasodilatory properties. Variability of the CYP2J2 gene is highly constrained except for its proximal promoter: there is a relatively common and functionally relevant single nucleotide polymorphism, indicated by -50G > T polymorphism (CYP2J2*7). Although genetic variation is known among ethnic groups, data for allele frequency are limited to a few Caucasian, Asian, and one African populations. In the present study, genotype distribution of CYP2J2*7 polymorphisms was investigated using polymerase chain reaction and restriction fragment length polymorphism assay in Japanese (n = 338), Mongolian (n = 118), and Ovambo (n = 186) populations and the findings compared with other populations. The mutant (CYP2J2*7) frequencies in the Japanese, Mongolians, and Ovambos were 0.0621, 0.0339, and 0.0672, respectively. Except for the Taiwanese, a general uniformity in the polymorphism in the Asian populations was observed. The mutation frequency of Ovambos was relatively lower than that of the African-American population. This study is the first to investigate the distribution of the CYP2J2*7 gene polymorphisms in Japanese, Mongolians, and Ovambos. These data will be informative and facilitate genetic association studies, in Asian and African populations for CYP2J2-related diseases such as cardiovascular disorders.

Cytochrome P450 (CYP) 2J2 gene transfection attenuates MMP‐9 via inhibition of NF‐κβ in hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase-9 (MMP-9). Cytochrome P450 (CYP) epoxygenase-2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF-kappabeta. However, the underlying molecular mechanisms for MMP-9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)-induced MMP-9 activation. CYP2J2 was over-expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF-kappabeta-mediated MMP-9 regulation were evaluated using Western blot, in-gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K-dependent AKT signal. Hcy induced the nuclear translocation of NF-kappabeta via downregulation of IKbetaalpha (endogenous cytoplasmic inhibitor of NF-kappabeta). Hcy induced MMP-9 activation by increasing NF-kappabeta-DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9-EET phosphorylated the AKT and attenuated Hcy-induced MMP-9 activation. This occurred, in part, by the inhibition of NF-kappabeta nuclear translocation, NF-kappabeta-DNA binding and activation of IKbetaalpha. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP-9 signal.

Genetic variation in cytochrome P450 2J2 and soluble epoxide hydrolase and risk of ischemic stroke in a Chinese population.

Epoxyeicosatrienoic acids have been recognized for their protective effects on the cardiovascular system. This study investigated whether two common polymorphisms in genes believed to be influential in regulating circulating levels of epoxyeicosatrienoic acids, namely cytochrome P450 2J2 (CYP2J2) G-50T and soluble epoxide hydrolase (EPHX2) G860A, were associated with ischemic stroke risk in a Chinese population. Screening of 200 patients with ischemic stroke and 350 control participants revealed that CYP2J2-50T allele frequency was not significantly different in ischemic stroke cases versus controls. In contrast, EPHX2 860A allele frequency was 16.8% in ischemic stroke cases versus 21.7% in controls (P=0.047), and the presence of this variant allele was associated with a significantly lower risk of ischemic stroke after adjustment for sex, age and multiple cardiovascular risk factors (adjusted odds ratio=0.50, 95% confidence interval 0.29-0.86). Moreover, there was a significant interaction between the EPHX2 G860A polymorphism, smoking and ischemic stroke risk such that nonsmokers carrying the EPHX2 G860A variant allele were at the lowest risk of ischemic stroke (odds ratio=0.33, 95% confidence interval, 0.17-0.67, P=0.002), whereas no significant association was observed in smokers. Collectively, these data indicate a protective influence of the G860A polymorphism of EPHX2 on ischemic stroke in Chinese nonsmokers.

A Common Polymorphism G‐50T in Cytochrome P450 2J2 Gene Is Associated with Increased Risk of Essential Hypertension in a Russian Population

The present study was designed to test whether common polymorphism G-50T within the promoter of human CYP2J2 gene is associated with increased risk of essential hypertension in a Russian population. We studied 576 unrelated subjects, including 295 patients with hypertension and 281 healthy subjects. Genotyping for polymorphism G-50T of the CYP2J2 gene was performed by polymerase chain reaction and restriction fragment length polymorphism techniques. The frequency of a −50T variant allele of CYP2J2 gene was significantly higher in patients with hypertension versus healthy controls (OR 4.03 95%CI 1.80–9.04 p=0.0004). The association of a −50GT genotype with hypertension remained significant after adjustment for age, gender and family history of hypertension by multivariate logistic regression (OR 4.78 95%CI 1.87–12.27 p=0.001). It has been found that OR for −50GT genotype × gender interaction (OR 4.48 95%CI 1.93–10.39 p=0.00048) was slightly higher than OR for −50GT genotype (OR 4.43 95%CI 1.91–10.29 p=0.00052), suggesting a weak effect of gender on the risk of hypertension in the heterozygous carriers of −50GT genotype. A family history of hypertension has no effect on the association between a −50GT genotype and hypertension. In present study we demonstrate for the first time that a CYP2J2*7 allele of the CYP2J2 gene is clearly associated with an increased risk of essential hypertension. Furthermore, this study highlights the importance of P-450 epoxygenase pathway of arachidonic acid metabolism in the pathogenesis of hypertensive disease.

Probing ligand binding modes of human cytochrome P450 2J2 by homology modeling, molecular dynamics simulation, and flexible molecular docking

Cytochrome P450 (P450) 2J2 catalyzes epoxidation of arachidonic acid to eicosatrienoic acids, which are related to a variety of diseases such as coronary artery disease, hypertension, and carcinogenesis. Recent experimental data also suggest that P450 2J2 could be a novel biomarker and a potential target for cancer therapy. However, the active site topology and substrate specificity of this enzyme remain unclear. In this study, a three-dimensional model of human P450 2J2 was first constructed on the basis of the crystal structure of human P450 2C9 in complex with a substrate using homology modeling method, and refined by molecular dynamics simulation. Flexible docking approaches were then employed to dock four ligands into the active site of P450 2J2 in order to probe the ligand-binding modes. By analyzing the results, active site architecture and certain key residues responsible for substrate specificity were identified on the enzyme, which might be very helpful for understanding the enzyme's biological role and providing insights for designing novel inhibitors of P450 2J2.

Promoter Polymorphism G-50T of a Human CYP2J2 Epoxygenase Gene Is Associated With Common Susceptibility to Asthma

Cytochrome P-450 2J2 (CYP2J2) has recently been shown to be an important enzyme in the metabolism of epoxygenase-derived eicosanoids that play important functional roles in pulmonary physiology and may contribute to the pathogenesis of asthma. The focus of our pilot study was to evaluate whether common polymorphism G-50T within the proximal promoter of human CYP2J2 gene is associated with the susceptibility to bronchial asthma. A total of 429 unrelated Russian subjects were recruited in this case-control study, including 215 sex-matched and age-matched patients with asthma and 214 healthy control subjects. The blood samples were analyzed for genetic polymorphism G-50T in the CYP2J2 gene by polymerase chain reaction followed by restriction fragment length polymorphism analysis. The frequency of variant allele -50T of the CYP2J2 gene was significantly higher in asthmatic patients than in healthy subjects (odds ratio [OR], 5.04; 95% confidence interval [CI], 1.99 to 12.77; p = 0.0003). In addition, the heterozygous genotype -50GT of the CYP2J2 gene was found to be significantly associated with susceptibility to allergic asthma (OR, 5.40; 95% CI, 2.05 to 14.26; p = 0.0003) as well as nonallergic asthma (OR, 5.77; 95% CI, 1.84 to 18.10; p = 0.004). The associations of the CYP2J2 gene G-50T polymorphism with asthma remained significant after adjustment for age and gender using multiple logistic regression analysis. Our data demonstrate for the first time that the CYP2J2 gene might be considered as a novel candidate gene for common susceptibility to asthma and highlight the importance of the P-450 epoxygenase pathway of metabolism of arachidonic acid in the pathogenesis of the disease.