Ghada Abdelhamid

Soluble epoxide hydrolase inhibitor, TUPS, protects against isoprenaline‐induced cardiac hypertrophy

We have previously shown that isoprenaline‐induced cardiac hypertrophy causes significant changes in the expression of cytochromes P450 (CYP) and soluble epoxide hydrolase (sEH) genes. Therefore, it is important to examine whether the inhibition of sEH by 1‐(1‐methanesulfonyl‐piperidin‐4‐yl)‐3‐(4‐trifluoromethoxy‐phenyl)‐urea (TUPS) will protect against isoprenaline‐induced cardiac hypertrophy.

Cytochrome P450 epoxygenase metabolite, 14,15-EET, protects against isoproterenol-induced cellular hypertrophy in H9c2 rat cell line.

We have previously shown that isoproterenol-induced cardiac hypertrophy causes significant changes to cytochromes P450 (CYPs) and soluble epoxide hydrolase (sEH) gene expression. Therefore, in this study, we examined the effect of isoproterenol in H9c2 cells, and the protective effects of 14,15-EET against isoproterenol-induced cellular hypertrophy. Isoproterenol was incubated with H9c2 cells for 24 and 48 h. To determine the protective effects of 14,15-EET, H9c2 cells were incubated with isoproterenol in the absence and presence of 14,15-EET. Thereafter, the expression of hypertrophic markers and different CYP genes were determined by real time-PCR. Our results demonstrated that isoproterenol significantly increased the expression of hypertrophic marker, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), parallel to a significant increase in cell surface area. Also, isoproterenol increased the mRNA expression of CYP1A1, CYP1B1, CYP2J3, CYP4F4 and CYP4F5, as well as the gene encoding sEH, EPHX2. On other hand, 14,15-EET significantly attenuated the isoproterenol-mediated induction of ANP, BNP, CYP1A1, CYP2J3, CYP4F4, CYP4F5 and EPHX2. Moreover 14,15-EET prevented the isoproterenol-mediated increase in cell surface area. Interestingly, 20-hydroxyeicosatetraenoic acid (20-HETE) treatment caused similar effects to that of isoproterenol treatment and induced cellular hypertrophy in H9c2 cells. In conclusion, isoproterenol induces cellular hypertrophy and modulates the expression of CYPs and EPHX2 in H9c2 cells. Furthermore, 14,15-EET exerts a protective effect against isoproterenol-induced cellular hypertrophy whereas, 20-HETE induced cellular hypertrophy in H9c2 cells.

Murine atrial HL-1 cell line is a reliable model to study drug metabolizing enzymes in the heart.

HL-1 cells are currently the only cells that spontaneously contract while maintaining a differentiated cardiac phenotype. Thus, our objective was to examine murine HL-1 cells as a new in vitro model to study drug metabolizing enzymes. We examined the expression of cytochrome P450s (Cyps), phase II enzymes, and nuclear receptors and compared their levels to mice hearts. Our results demonstrated that except for Cyp4a12 and Cyp4a14 all Cyps, phase II enzymes: glutathione-S-transferases (Gsts), heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase (Nqo1), nuclear receptors: aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator activated receptor (PPAR-alpha) were all constitutively expressed in HL-1 cells. Cyp2b19, Cyp2c29, Cyp2c38, Cyp2c40, and Cyp4f16 mRNA levels were higher in HL-1 cells compared to mice hearts. Cyp2b9, Cyp2c44, Cyp2j9, Cyp2j11, Cyp2j13, Cyp4f13, Cyp4f15 mRNA levels were expressed to the same extent to that of mice hearts. Cyp1a1, Cyp1a2, Cyp1b1, Cyp2b10, Cyp2d10, Cyp2d22, Cyp2e1, Cyp2j5, Cyp2j6, Cyp3a11, Cyp4a10, and Cyp4f18 mRNA levels were lower in HL-1 cells compared to mice hearts. Moreover, 3-methylcholanthrene induced Cyp1a1 while fenofibrate induced Cyp2j9 and Cyp4f13 mRNA levels in HL-1 cells. Examining the metabolism of arachidonic acid (AA) by HL-1 cells, our results demonstrated that HL-1 cells metabolize AA to epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids, and 20-hydroxyeicosatetraenoic acids. In conclusion, HL-1 cells provide a valuable in vitro model to study the role of Cyps and their associated AA metabolites in addition to phase II enzymes in cardiovascular disease states.

Human fetal ventricular cardiomyocyte, RL-14 cell line, is a promising model to study drug metabolizing enzymes and their associated arachidonic acid metabolites

INTRODUCTION RL-14 cells, human fetal ventricular cardiomyocytes, are a commercially available cell line that has been established from non-proliferating primary cultures derived from human fetal heart tissue. However, the expression of different drug metabolizing enzymes (DMEs) in RL-14 cells has not been elucidated yet. Therefore, the main objectives of the current work were to investigate the capacity of RL-14 cells to express different cytochrome P450 (CYP) isoenzymes and correlate this expression to primary cardiomyocytes. METHODS The expression of CYP isoenzymes was determined at mRNA, protein and catalytic activity levels using real time-PCR, Western blot analysis and liquid chromatography-electron spray ionization-mass spectrometry (LC-ESI-MS), respectively. RESULTS Our results showed that RL-14 cells constitutively express CYP ω-hydroxylases, CYP1A, 1B, 4A and 4F; CYP epoxygenases, CYP2B, 2C and 2J; in addition to soluble epoxide hydrolayse (EPHX2) at mRNA and protein levels. The basal expression of CYP ω-hydroxylases, epoxygenases and EPHX2 was supported by the ability of RL-14 cells to convert arachidonic acid to its biologically active metabolites, 20-hydroxyeicosatetraenoic acids (20-HETEs), 14,15-epoxyeicosatrienoic acids (14,15-EET), 11,12-EET, 8,9-EET, 5,6-EET, 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), 11,12-DHET, 8,9-DHET and 5,6-DHET. Furthermore, RL-14 cells express CYP epoxygenases and ω-hydroxylase at comparable levels to those expressed in adult and fetal human primary cardiomyocytes cells implying the importance of RL-14 cells as a model for studying DMEs in vitro. Lastly, different CYP families were induced in RL-14 cells using 2,3,7,8-tetrachlorodibenzo-p-dioxin and fenofibrate at mRNA and protein levels. DISCUSSION The current study provides the first evidence that RL-14 cells express CYP isoenzymes at comparable levels to those expressed in the primary cells and thus offers a unique in vitro model to study DMEs in the heart.

Transcriptional and posttranscriptional regulation of CYP1A1 by vanadium in human hepatoma HepG2 cells

We recently demonstrated that V5+ downregulates 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of Cyp1a1 mRNA, protein, and catalytic activity levels in Hepa 1c1c7 cells through transcriptional mechanism. Therefore, it is important to investigate whether similar changes occur in humans. For this purpose, we examined the effect of V5+ (as ammonium metavanadate, NH4VO3) on the expression of aryl hydrocarbon receptor (AhR)-regulated gene; cytochrome P450 1A1 (CYP1A1) at each step of the AhR signal transduction pathway in human hepatoma HepG2 cells. Our results show a significant reduction in TCDD-mediated induction of CYP1A1 mRNA, protein, and activity levels after V5+ treatment in a dose-dependent manner. Investigating the effect of co-exposure to V5+ and TCDD at transcriptional levels revealed that V5+ significantly inhibited TCDD-mediated induction of AhR-dependent luciferase reporter gene expression. Looking at the posttranscriptional level, V5+ did not affect CYP1A1 mRNA stability, thus eliminating the possible role of V5+ in modifying CYP1A1 gene expression through this mechanism. On the other hand, at the posttranslational level, V5+ was able to significantly decrease CYP1A1 protein half-life contributing to the inconsistency between catalytic activity and transcriptional level. Importantly, we showed that V5+ did not significantly alter the heme oxygenase-1 mRNA level, thus eliminating any possibility that V5+ might have decreased CYP1A1 activity through affecting its heme content. This study demonstrates for the first time that V5+ downregulates the expression of CYP1A1 at the transcriptional, posttranscriptional and posttranslational mechanisms in the human hepatoma HepG2 cells.

Buthionine sulfoximine, an inhibitor of glutathione biosynthesis, induces expression of soluble epoxide hydrolase and markers of cellular hypertrophy in a rat cardiomyoblast cell line: Roles of the NF-κB and MAPK signaling pathways

Evidence suggests that upregulation of soluble epoxide hydrolase (sEH) is associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac hypertrophy, and heart failure. However, the upregulation mechanism is still unknown. In this study, we treated H9C2 cells with buthionine sulfoximine (BSO) to explore whether oxidative stress upregulates sEH gene expression and to identify the molecular and cellular mechanisms behind this upregulatory response. Real-time PCR and Western blot analyses were used to measure mRNA and protein expression, respectively. We demonstrated that BSO significantly upregulated sEH at mRNA levels in a concentration- and time-dependent manner, leading to a significant increase in the cellular hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Furthermore, BSO significantly increased the cytosolic phosphorylated IκB-α and translocation of NF-κB p50 subunits, as measured by Western blot analysis. This level of translocation was paralleled by an increase in the DNA-binding activity of NF-κB P50 subunits. Moreover, our results demonstrated that pretreatment with the NF-κB inhibitor PDTC significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression in a dose-dependent manner. Additionally, mitogen-activated protein kinases (MAPKs) were transiently phosphorylated by BSO treatment. To understand further the role of MAPKs pathway in BSO-mediated induction of sEH mRNA, we examined the role of extracellular signal-regulated kinase (ERK), c-JunN-terminal kinase (JNK), and p38 MAPK. Indeed, treatment with the MEK/ERK signal transduction inhibitor, PD98059, partially blocked the activation of IκB-α and translocation of NF-κB p50 subunits induced by BSO. Moreover, pretreatment with MEK/ERK signal transduction inhibitors, PD98059 and U0126, significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression. These results clearly demonstrated that the NF-κB signaling pathway is involved in BSO-mediated induction of sEH gene expression, and appears to be associated with the activation of the MAPK pathway. Furthermore, our findings provide a strong link between sEH-induced cardiac dysfunction and involvement of NF-κB in the development of cellular hypertrophy.

Development of cellular hypertrophy by 8-hydroxyeicosatetraenoic acid in the human ventricular cardiomyocyte, RL-14 cell line, is implicated by MAPK and NF-κB

Recent studies have established the role of mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) in the development of cardiovascular disease. Among these mid-chains, 8-HETE has been reported to have a proliferator and proinflammatory action. However, whether 8-HETE can induce cardiac hypertrophy has never been investigated before. Therefore, the overall objectives of the present study are to elucidate the potential hypertrophic effect of 8-HETE in the human ventricular cardiomyocytes, RL-14 cells, and to explore the mechanism(s) involved. Our results showed that 8-HETE induced cellular hypertrophy in RL-14 cells as evidenced by the induction of cardiac hypertrophy markers ANP, BNP, α-MHC, and β-MHC in a concentration- and time-dependent manner as well as the increase in cell surface area. Mechanistically, 8-HETE was able to induce the NF-κB activity as well as it significantly induced the phosphorylation of ERK1/2. The induction of cellular hypertrophy was associated with a proportional increase in the formation of dihydroxyeicosatrienoic acids (DHETs) parallel to the increase of soluble epoxide hydrolase (sEH) enzyme activity. Blocking the induction of NF-κB, ERK1/2, and sEH signaling pathways significantly inhibited 8-HETE-induced cellular hypertrophy. Our study provides the first evidence that 8-HETE induces cellular hypertrophy in RL-14 cells through MAPK- and NF-κB-dependent mechanism.

Modulation of NAD(P)H:quinone oxidoreductase by vanadium in human hepatoma HepG2 cells.

Recent studies demonstrated the carcinogenicity and the mutagenicity of vanadium compounds. In addition, vanadium (V(5+)) was found to enhance the effects of other genotoxic agents. However, the mechanism by which V(5+) induce toxicity remain unknown. In the current study we examined the effect of V(5+) (as ammonium metavanadate, NH(4)VO(3)) on the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) in human hepatoma HepG2 cells. Therefore, HepG2 cells were treated with increasing concentrations of V(5+) in the presence of two NQO1 inducers, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL). Our results showed that V(5+) inhibited the TCDD- and SUL-mediated induction of NQO1 at mRNA, protein and activity levels. Investigating the effect of V(5+) at transcriptional levels revealed that V(5+) significantly inhibited the TCDD- and SUL-mediated induction of antioxidant responsive element (ARE)-dependent luciferase reporter gene expression. In addition, V(5+) was able to decrease the TCDD- and SUL-induced nuclear accumulation of nuclear factor erythroid 2-related factor-2 (Nrf2) without affecting Nrf2 mRNA or protein levels. Looking at the post-transcriptional level, V(5+) did not affect NQO1 mRNA stability, thus eliminating the possible role of V(5+) in decreasing NQO1 mRNA levels through this mechanism. In contrast, at post-translational level, V(5+) was able to significantly decrease NQO1 protein half-life. The present study demonstrates for the first time that V(5+) down-regulates NQO1 at the transcriptional and post-translational levels in the human hepatoma HepG2 cells via AhR- and Nrf2-dependent mechanisms.

19-Hydroxyeicosatetraenoic acid and isoniazid protect against angiotensin II-induced cardiac hypertrophy.

We have recently demonstrated that 19-hydroxyeicosatetraenoic acid (19-HETE) is the major subterminal-HETE formed in the heart tissue, and its formation was decreased during cardiac hypertrophy. In the current study, we examined whether 19-HETE confers cardioprotection against angiotensin II (Ang II)-induced cardiac hypertrophy. The effect of Ang II, with and without 19-HETE (20 μM), on the development of cellular hypertrophy in cardiomyocyte RL-14 cells was assessed by real-time PCR. Also, cardiac hypertrophy was induced in Sprague-Dawley rats by Ang II, and the effect of increasing 19-HETE by isoniazid (INH; 200mg/kg/day) was assessed by heart weight and echocardiography. Also, alterations in cardiac cytochrome P450 (CYP) and their associated arachidonic acid (AA) metabolites were determined by real-time PCR, Western blotting and liquid-chromatography-mass-spectrometry. Our results demonstrated that 19-HETE conferred a cardioprotective effect against Ang II-induced cellular hypertrophy in vitro, as indicated by the significant reduction in β/α-myosin heavy chain ratio. In vivo, INH improved heart dimensions, and reversed the increase in heart weight to tibia length ratio caused by Ang II. We found a significant increase in cardiac 19-HETE, as well as a significant reduction in AA and its metabolite, 20-HETE. In conclusion, 19-HETE, incubated with cardiomyocytes in vitro or induced in the heart by INH in vivo, provides cardioprotection against Ang II-induced hypertrophy. This further confirms the role of CYP, and their associated AA metabolites in the development of cardiac hypertrophy.

Effect of mercury on aryl hydrocarbon receptor-regulated genes in the extrahepatic tissues of C57BL/6 mice

The individual toxic effects of aryl hydrocarbon receptors (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or heavy metals typified by mercury (Hg(2+)) has been previously demonstrated. However, little is known about the combined toxic effects of TCDD and Hg(2+)in vivo. Therefore, we examined the effect of exposure to Hg(2+) (2.5mg/kg) in the absence and presence of TCDD (15 μg/kg) on the AhR-regulated genes using C57Bl/6 mice. Hg(2+) alone did not affect kidney, lung, or heart Cyp1a1/1a2/1b1 mRNA levels. On the contrary, Hg(2+) alone significantly induced kidney Cyp1a1/1a2/1b1 and lung Cyp1b1 protein and catalytic activities. Hg(2+) also induced Nqo1, Gsta1, and HO-1 at the mRNA, protein, and activity levels in the kidney and heart but not in the lung. Upon co-exposure to Hg(2+) and TCDD, Hg(2+) significantly potentiated the TCDD-mediated induction of kidney and lung Cyp1a1/1a2/1b1 mRNA levels, while it decreased their kidney protein and catalytic activity and it increased their lung protein. In addition, Hg(2+) potentiated the TCDD-mediated induction of Nqo1, Gsta1, and HO-1 at mRNA, protein and activity levels in all tissues. The present study demonstrates that Hg(2+) modulates the constitutive and TCDD-induced AhR-regulated genes in a time-, tissue- and, AhR-regulated enzyme genes manner.