The Role of Soluble Epoxide Hydrolase Enzyme on Daunorubicin-Mediated Cardiotoxicity.

Several studies have demonstrated the role of cytochrome P450 (CYP) and its associated arachidonic acid (AA) metabolites in the anthracyclines-induced cardiac toxicity. However, the ability of daunorubicin (DNR) to induce cardiotoxicity through the modulation of CYP and its associated AA metabolites has not been investigated yet. Therefore, we hypothesized that DNR-induced cardiotoxicity is mediated through the induction of cardiotoxic hydroxyeicosatetraenoic acids and/or the inhibition of cardioprotctive epoxyeicosatrienoic acids (EETs). To test our hypothesis, Sprague-Dawley rats were treated with DNR (5 mg/kg i.p.) for 24 h, whereas human ventricular cardiomyocytes RL-14 cells were exposed to DNR in the presence and absence of 4-[[trans-4-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]cyclohexyl]oxy]-benzoic acid (tAUCB), a soluble epoxide hydrolase (sEH) inhibitor. Thereafter, real-time PCR, Western blot analysis and liquid chromatography-electron spray ionization mass spectroscopy were used to determine the level of gene expression, protein expression and AA metabolites, respectively. Our results showed that DNR-induced cardiotoxicity in vivo and in vitro as evidenced by the induction of hypertrophic and fibrotic markers. Moreover, the DNR-induced cardiotoxicity was associated with a dramatic increase in the formation of cardiac DHET/EET metabolites both in vivo and in RL-14 cells suggesting a sEH enzyme dependent mechanism. Interestingly, inhibition of sEH using tAUCB, a selective sEH inhibitor, significantly protects against DNR-induced cardiotoxicity. Mechanistically, the protective effect tAUCB was mediated through the induction of P50 nuclear factor-κB and the inhibition of phosphorylated p38. In conclusion, our study provides the first evidence that DNR induces cardiotoxicity through a sEH-mediated EETs degradation-dependent mechanism.

Dimethylarsinic acid modulates the aryl hydrocarbon receptor-regulated genes in C57BL/6 mice: in vivo study.

1. Dimethylarsinic acid (DMA(V)) is the major metabolite of inorganic arsenic in human body. Thus we investigated the effect of DMA(V) on the alteration of phase I (typified by Cyp1a) and phase II (typified by Nqo1) AhR-regulated genes in vivo. C57BL/6 mice received DMA(V) (13.3 mg/kg, i.p.) with or without TCDD (15 µg/kg, i.p.), thereafter the liver, lung, and kidney were harvested at 6 and 24 h post-treatment. 2. Results demonstrated that DMA(V) has no significant effect on Cyp1a mRNA and protein expression or catalytic activity in the liver. On the other hand, DMA(V) significantly potentiated the TCDD-mediated induction of Cyp1a mRNA and protein expression, with a subsequent potentiation of catalytic activity in the lung. Moreover, DMA(V) significantly inhibited the TCDD-mediated induction of Cyp1a mRNA and protein expression with subsequent inhibition of catalytic activity in the kidney. 3. Regarding to phase II AhR-regulated genes, DMA(V) has no significant effect on Nqo1 mRNA and protein expression, or activity neither in the liver, lung, or kidney. 4. In conclusion, the present work demonstrates for the first time that DMA(V) modulates AhR-regulated genes in a tissue- and enzyme-specific manner. This modulation may play a crucial role in arsenic-induced toxicity and carcinogenicity.

Inhibition of Mid-chain HETEs Protects Against Angiotensin II-induced Cardiac Hypertrophy.

Recent data demonstrated the role of CYP1B1 in cardiovascular disease. It was, therefore, necessary to examine whether the inhibition of CYP1B1 and hence inhibiting the formation of its metabolites, using 2,4,3',5'-tetramethoxystilbene (TMS), would have a cardioprotective effect against angiotensin II (Ang II)-induced cardiac hypertrophy. For this purpose, male Sprague Dawley rats were treated with Ang II with or without TMS (300 µg/kg every third day i.p.). Thereafter, cardiac hypertrophy and the formation of mid-chain HETEs and arachidonic acid were assessed. In vitro, RL-14 cells were treated with Ang II (10 µM) in the presence and absence of TMS (0.5 µM). Then, reactive oxygen species, mitogen-activated protein kinase phosphorylation levels, and nuclear factor-kappa B-binding activity were determined. Our results demonstrated that TMS protects against Ang II-induced cardiac hypertrophy as indicated by the improvement in cardiac functions shown by the echocardiography as well as by reversing the increase in heart weight to tibial length ratio caused by Ang II. In addition, the cardioprotective effect of TMS was associated with a significant decrease in cardiac mid-chain HETEs levels. Mechanistically, TMS inhibited reactive oxygen species formation, the phosphorylation of ERK1/2, p38 mitogen-activated protein kinase, and the binding of p65 NF-κB.

Clinical Implications of 20-Hydroxyeicosatetraenoic Acid in the Kidney, Liver, Lung and Brain: An Emerging Therapeutic Target.

Cytochrome P450-mediated metabolism of arachidonic acid (AA) is an important pathway for the formation of eicosanoids. The ω-hydroxylation of AA generates significant levels of 20-hydroxyeicosatetraenoic acid (20-HETE) in various tissues. In the current review, we discussed the role of 20-HETE in the kidney, liver, lung, and brain during physiological and pathophysiological states. Moreover, we discussed the role of 20-HETE in tumor formation, metabolic syndrome and diabetes. In the kidney, 20-HETE is involved in modulation of preglomerular vascular tone and tubular ion transport. Furthermore, 20-HETE is involved in renal ischemia/reperfusion (I/R) injury and polycystic kidney diseases. The role of 20-HETE in the liver is not clearly understood although it represents 50%-75% of liver CYP-dependent AA metabolism, and it is associated with liver cirrhotic ascites. In the respiratory system, 20-HETE plays a role in pulmonary cell survival, pulmonary vascular tone and tone of the airways. As for the brain, 20-HETE is involved in cerebral I/R injury. Moreover, 20-HETE has angiogenic and mitogenic properties and thus helps in tumor promotion. Several inhibitors and inducers of the synthesis of 20-HETE as well as 20-HETE analogues and antagonists are recently available and could be promising therapeutic options for the treatment of many disease states in the future.

The Obesogenic Potency of Various High-Caloric Diet Compositions in Male Rats, and Their Effects on Expression of Liver and Kidney Proteins Involved in Drug Elimination.

Obesity is caused by a number of factors including heredity, lack of exercise, and poor diet. Diets rich in fats and carbohydrates are the common culprits leading to obesity. Here we studied the effects of these components on proteins involved in drug disposition. Male rats were given a normal diet (lean controls) or one rich in fats, carbohydrates (as high-fructose corn syrup equivalent) or in combination. After 14 weeks, plasma biochemistry, liver and kidney mRNA and protein for selected cytochrome P450 (CYP) and transporters were determined. Significant increases in body and perinephric fat weight were noted in each of the high-calorie diet-fed groups, with increases being higher in those given high-fat diets. Increases in the protein of CYP3A1/2 and CYP2C11 were seen in liver in high-fat-fed rats. No changes were seen for CYP1A1 at the level of mRNA or protein. For transporters, decreases in expressions of Oct1/2 and Mate1 were seen, with no change in Mdr1. The results showed similarity to earlier assessments of genetically prone rats and suggested that diet-induced obesity has the potential to lead to decreases in the clearance of drugs acting as substrates for CYP 3A, 2C11, and organic cation transport.

Down-regulation of cytochrome P450 1A1 by monomethylarsonous acid in human HepG2 cells.

Inorganic arsenic is a human toxicant and carcinogen that has been extensively studied over decades; however, no definitive understanding of the underlying mechanisms has been established yet. Arsenic is capable of modulating the expression of aryl hydrocarbon receptor (AhR)-regulated genes, nevertheless, whether its trivalent organic metabolites have similar effects or not need to be investigated. Therefore, in this study we examined the effects of monomethylarsonous acid (MMA(III)) as compared to its parent compound sodium arsenite (As(III)) on the expression of CYP1A1 in HepG2 cells. HepG2 cells were treated with MMA(III) (5µM) or its parents compound, As(III) (5µM), in the absence and presence of the prototypical AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1nM). Experiments were conducted at 6h for gene expression; 24h for XRE-driven luciferase activity, protein expression, and EROD activity. Our results showed that both MMA(III) and As(III) decreased CYP1A1 mRNA, protein, and catalytic activity levels; and inhibit the TCDD-mediated induction of CYP1A1 mRNA, protein, and catalytic activity levels. MMA(III) and As(III) significantly inhibited XRE-driven luciferase activity and it inhibited the TCDD-mediated induction of XRE-driven luciferase reporter gene expression. Although MMA(III) and As(III) were not shown to be AhR ligands, both compounds showed inhibition of nuclear accumulation of AhR transcription factor as evidenced by immunocytochemical analysis. MMA(III) and As(III) had no effect on CYP1A1 mRNA stability; however MMA(III), but not As(III), decreased the protein stability of CYP1A1. As(III), but not MMA(III), induced HO-1 mRNA levels. Both MMA(III) and As(III) increased ROS production. Our results demonstrate for the first time that, MMA(III) down-regulates CYP1A1 mainly through transcriptional and post-translational mechanisms. This modulation of CYP1A1 proves that trivalent metabolites of arsenic are highly reactive and could participate in arsenic toxicity.

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 ß/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.

Modulation of aryl hydrocarbon receptor-regulated enzymes by trimethylarsine oxide in C57BL/6 mice: In vivo and in vitro studies.

Arsenic is a worldwide environmental pollutant that is associated with skin and several types of internal cancers. Recent reports revealed that arsenic biomethylation could activate the toxic and carcinogenic potential of arsenic. Therefore, we investigated the effect of trimethylarsine oxide (TMAO) on the activation of AhR-regulated genes in vivo and in vitro. In vivo, C57BL/6 mice received TMAO (13mg/kg i.p.) with or without the prototypical AhR ligand, TCDD (15µg/kg), then the livers were harvested at 6 and 24h post-treatment. In vitro, isolated hepatocytes from C57BL/6 mice were treated with TMAO (5µM) in the absence and presence of TCDD (1nM) for 6 and 24h. Our in vivo results demonstrated that, TMAO alone increased Cyp1a1, Cyp1a2, Cyp1b1, Nqo1, Gsta1, and Ho-1 at mRNA level. Upon co-exposure to TMAO and TCDD, TMAO potentiated the TCDD-mediated induction of Cyp1a1, Cyp1b1, and Nqo1 mRNA levels. Western blotting revealed that, TMAO alone increased Cyp1a1, Cyp1a2, Nqo1, Gsta1/2, and Ho-1 protein levels, and potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 protein level. In addition, TMAO alone significantly increased Cyp1a1, Cyp1a2, Nqo1, Gst, and Ho-1 activities and significantly potentiated the TCDD-mediated induction of Cyp1a1 activity. At the in vitro level, TMAO induced Cyp1a1 and potentiated the TCDD-mediated induction of Cyp1a1 at mRNA, protein and activity levels. In addition, TMAO increased the nuclear localization of AhR and AhR-dependent XRE-driven luciferase activity. Our results demonstrate that the TMAO, modulates AhR-regulated genes which could potentially participate, at least in part, in arsenic induced toxicity and carcinogenicity.

Modulation of aryl hydrocarbon receptor regulated genes by acute administration of trimethylarsine oxide in the lung, kidney and heart of C57BL/6 mice.

1. Arsenite alters the expression of aryl hydrocarbon receptor (AhR)-regulated genes in extrahepatic tissues; yet, the effect of organic arsenicals still unknown. Therefore, C57BL/6 mice received trimethylarsine oxide (TMAO; 13 mg/kg i.p.) with or without 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 15 µg/kg), and euthanized at 6 or 24 h. 2. Our results demonstrated that TMAO increased Cyp1a1 and Cyp1b1 mRNA, protein and activity in the lung. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1a2 mRNA, protein and activity in the lung. In the kidney, TMAO increased Cyp1b1 mRNA and protein. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA, protein and activity. In the heart, TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA. 3. Moreover, TMAO induced Nqo1 mRNA in the lung, kidney and heart, with subsequent increase in Nqo1 protein and activity in the lung. TMAO increased Gsta mRNA in the heart; and increased Gsta protein and activity in the lung and kidney. TMAO increased Nqo1 mRNA as compared to TCDD in the kidney and heart, and potentiated the TCDD-mediated induction of Gsta protein and activity in the kidney. 4. In conclusion, TMAO modulates AhR-regulated genes in a tissue- and enzyme-specific manner.

Early Changes in Cytochrome P450s and Their Associated Arachidonic Acid Metabolites Play a Crucial Role in the Initiation of Cardiac Hypertrophy Induced by Isoproterenol.

Cytochrome P450 enzymes (P450s), along with their cardioprotective metabolites the epoxyeicosatrienoic acids (EETs) and cardiotoxic metabolite 20-hydroxyeicosatetraeonic acid (20-HETE), were found to be altered in cardiac hypertrophy; however, it is unclear whether these changes are causal or epiphenomenon. Therefore, we hypothesized that P450s and their metabolites play a crucial role in the initiation of cardiac hypertrophy. To test our hypothesis, rats and RL-14 cells were treated with the hypertrophic agonist isoproterenol for different time periods. Thereafter, in vivo heart function and wall thickness were assessed using echocardiography. Moreover, the role of P450 epoxygenases, ω-hydroxylases, and soluble epoxide hydrolase (sEH) were determined at mRNA, protein, and activity levels using real-time polymerase chain reaction, Western blot, and liquid chromatography-mass spectrometry, respectively. Our results show that in vivo and in vitro hypertrophy was initiated after 72 hours and 6 hours of isoproterenol treatment, respectively. Studies performed at the prehypertrophy phase showed a significant decrease in P450 epoxygenases along with a significant induction of sEH activity. Consequently, lower EET and higher dihydroxyeicosatrienoic acid levels were observed during this phase. However, significant increases in P450 ω-hydroxylase along with its associated metabolite, 20-HETE, were detected only in vivo. Interestingly, increasing EET levels by P450 epoxygenase induction, sEH inhibition, or exogenous administration of EET prevented the initiation of cardiac hypertrophy through a nuclear factor-κB-mediated mechanism. Taken together, these findings reveal a crucial role of P450 epoxygenases and EETs in the development of cardiac hypertrophy, which could uncover novel targets for prevention of heart failure at early stages.

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.

Design and synthesis of resveratrol-salicylate hybrid derivatives as CYP1A1 inhibitors.

Resveratrol and aspirin are known to exert potential chemopreventive effects through modulation of numerous targets. Considering that the CYP450 system is responsible for the activation of environmental procarcinogens, the aim of this study was to design a new class of hybrid resveratrol-aspirin derivatives possessing the stilbene and the salicylate scaffolds. Using HepG2 cells, we evaluated (a) the inhibition of TCDD-mediated induction of CYP1A1 exerted by resveratrol-aspirin derivatives using the EROD assay, and (b) CYP1A1 mRNA in vitro. We observed significant inhibition (84%) of CYP1A1 activity and a substantial decrease in CYP1A1 mRNA with compound 3, compared to control. Resveratrol did not exert inhibition under the same experimental conditions. This inhibitory profile was supported by docking studies using the crystal structure of human CYP1A1. The potential effect exerted by compound 3 (the most active), provide preliminary evidence supporting the design of hybrid molecules combining the chemical features of resveratrol and aspirin.

Acute arsenic treatment alters arachidonic acid and its associated metabolite levels in the brain of C57Bl/6 mice.

The toxic effects of arsenic on the whole brain, as well as the discrete regions, has been previously reported for mice. We investigated the effects of acute arsenite (As(III)) on brain levels of arachidonic acid (AA) and its associated metabolites generated through cytochrome P450 (CYP), cyclooxygenase (COX), and lipoxygenase (LOX) pathways. Our results demonstrated that acute As(III) treatment (12.5 mg·(kg body mass)(-1)) decreases cytosolic phospholipase A2 (cPLA2) with a subsequent decrease in its catalytic activity and brain AA levels. In addition, As(III) differentially altered CYP epoxygenases and CYP ω-hydroxylases, but it did not affect brain Ephx2 mRNA or sEH catalytic activity levels. As(III)-mediated effects on Cyps caused an increase in brain 5,6-epoxyeicosatrienoic acid (5,6-EET) and 16/17-hydroxyeicosatetreinoic acid (16/17-HETE) levels, and a decrease in 18- and 20-HETE levels. Furthermore, As(III) increased cyclooxygenase-2 (COX-2) mRNA while decreasing prostaglandins F2α (PGF2α) and PGJ2. As(III) also increased brain 5-lipoxygenase (5-LOX) and 15-LOX mRNA, but decreased 12-LOX mRNA. These changes in LOX mRNA were associated with a decrease in 8/12-HETE levels only. In conclusion, this is the first demonstration that As(III) decreases AA levels coinciding with alterations to EET, HETE, and PG levels, which affects brain development and neurochemistry.

Acute mercury toxicity modulates cytochrome P450, soluble epoxide hydrolase and their associated arachidonic acid metabolites in C57Bl/6 mouse heart.

Mercury exposure is associated with increased risk of cardiovascular disease and profound cardiotoxicity. However, the correlation between Hg(2+)-mediated toxicity and alteration in cardiac cytochrome P450s (Cyp) and their dependent arachidonic acid metabolites has never been investigated. Therefore, we investigated the effect of acute mercury toxicity on the expression of Cyp-epoxygenases and Cyp-ω-hydroxylases and their associated arachidonic acid metabolites in mice hearts. In addition, we examined the expression and activity of soluble epoxide hydrolase (sEH) as a key player in arachidonic acid metabolism pathway. Mercury toxicity was induced by a single intraperitoneal injection (IP) of 2.5 mg/kg of mercuric chloride (HgCl2). Our results showed that mercury treatment caused a significant induction of the cardiac hypertrophy markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP); in addition to Cyp1a1, Cyp1b1, Cyp2b9, Cyp2b10, Cyp2b19, Cyp2c29, Cyp2c38, Cyp4a10, Cyp4a12, Cyp4a14, Cyp4f13, Cyp4f15, Cyp4f16 and Cyp4f18 gene expression. Moreover, Hg(2+) significantly increased sEH protein expression and activity levels in hearts of mercury-treated mice, with a consequent decrease in 14,15-, and 11,12-epoxyeicosatrienoic acids (EETs) levels. Whereas the formation of 14,15-, 11,12-, 8,9-dihydroxyeicosatrienoic acids (DHETs) was significantly increased. In conclusion, acute Hg(2+) toxicity modulates the expression of several Cyp and sEH enzymes with a consequent decrease in the cardioprotective EETs which could represent a novel mechanism by which mercury causes progressive cardiotoxicity. Furthermore, inhibiting sEH might represent a novel therapeutic approach to prevent Hg(2+)-induced hypertrophy.

Fenofibrate modulates cytochrome P450 and arachidonic acid metabolism in the heart and protects against isoproterenol-induced cardiac hypertrophy.

It has been previously shown that the cytochrome P450 (P450) modulator, fenofibrate, protects against cardiovascular diseases. P450 and their metabolites, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) were found to play an important role in cardiovascular diseases. Therefore, it is important to examine whether fenofibrate would modulate the cardiac P450 and its associated arachidonic acid metabolites and whether this modulation protects against isoproterenol-induced cardiac hypertrophy. For this purpose, male Sprague-Dawley rats were treated with fenofibrate (30 mg·kg·d), isoproterenol (4.2 mg·kg·d), or the combination of both. The expression of hypertrophic markers and different P450s along with their metabolites was determined. Our results showed that fenofibrate significantly induced the cardiac P450 epoxygenases, such as CYP2B1, CYP2B2, CYP2C11, and CYP2C23, whereas it decreased the cardiac ω-hydroxylase, CYP4A3. Moreover, fenofibrate significantly increased the formation of 14,15-EET, 11,12-EET, and 8,9-EET, whereas it decreased the formation of 20-HETE in the heart. Furthermore, fenofibrate significantly decreased the hypertrophic markers and the increase in heart-to-body weight ratio induced by isoproterenol. This study demonstrates that fenofibrate alters the expression of cardiac P450s and their metabolites and partially protects against isoproterenol-induced cardiac hypertrophy, which further confirms the role of P450s, EETs, and 20-HETE in the development of cardiac hypertrophy.

Methylated pentavalent arsenic metabolites are bifunctional inducers, as they induce cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase through AhR- and Nrf2-dependent mechanisms.

Activation of the aryl hydrocarbon receptor (AhR) ultimately leads to the induction of the carcinogen-activating enzyme cytochrome P450 1A1 (CYP1A1), and activation of the nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) in addition to the AhR pathway induces the expression of the NADP(H):quinone oxidoreductase (NQO1). Therefore, the aim of this study was to examine the effect of As(III) pentavalent metabolites, MMA(V), DMA(V), and TMA(V), on AhR and Nrf2 activation and on the expression of their prototypical downstream targets CYP1A1 and NQO1, respectively. Our results showed that treatment of HepG2 cells with MMA(V), DMA(V), or TMA(V) in the absence and presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin or sulforaphane significantly induced both CYP1A1 and NQO1 at the mRNA, protein, and catalytic activity levels. Furthermore, these metabolites increased the AhR-dependent XRE-driven and the Nrf2-dependent ARE-driven luciferase reporter activities, which coincided with increased nuclear accumulation of both transcription factors. However, none of these metabolites were shown to be AhR ligands. The induction of CYP1A1 by these metabolites seems to be ligand-independent, possibly through a decrease in HSP90 protein expression levels. The metabolites also increased ROS production, which was significantly higher than that produced by As(III). Upon knockdown of AhR and Nrf2 the MMA(V)-, DMA(V)-, and TMA(V)-mediated induction of both CYP1A1 and NQO1 proteins was significantly decreased. In conclusion, this study demonstrates for the first time that methylated pentavalent arsenic metabolites are bifunctional inducers, as they increase CYP1A1 by activating the AhR/XRE signaling pathway and they increase NQO1 by activating the Nrf2/ARE signaling pathway in addition to the AhR/XRE pathway.

20-Hydroxyeicosatetraenoic acid is a potential therapeutic target in cardiovascular diseases.

Arachidonic acid (AA) is metabolized by enzymes of the cytochrome P450 (CYP) 4A and CYP4F subfamilies to 20- hydroxyeicosatetraeonic acid (20-HETE), which plays an important role in the cardiovascular system. In the current work, we reviewed the formation of 20-HETE in different species by different CYPs; 20-HETE metabolism by cyclooxygenases (COXs) and different isomerases; and the current available inducers and inhibitors of 20-HETE formation in addition to its agonists and antagonists. Moreover we reviewed the negative role of 20-HETE in cardiac hypertrophy, cardiotoxicity, diabetic cardiomyopathy, and in ischemia/reperfusion (I/R) injury. Lastly, we reviewed the role of 20-HETE in different hypertension models such as the renin/angiotensin II model, Goldblatt model, spontaneously hypertensive rat model, androgen-induced model, slat- and deoxycorticosterone acetate (DOCA)-salt-induced models, and high fat diet model. 20-HETE can affect pro- and anti-hypertensive mechanisms dependent upon where, when, and by which isoform it has been produced. In contrast to hypertension we also reviewed the role of 20-HETE in endotoxin-induced hypotension and the natriuretic effects of 20-HETE. Based on the recent studies, 20-HETE production and/or action might be a therapeutic target to protect against the initiation and progression of cardiovascular diseases.

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.

Acute arsenic treatment alters cytochrome P450 expression and arachidonic acid metabolism in lung, liver and kidney of C57Bl/6 mice.

1. Arsenic (As(III)) toxicity has received increasing attention as human exposure to arsenic is associated with pulmonary, hepatic and renal toxicities. Therefore, in the present study, we investigated the effect of acute As(III) treatment on pulmonary, hepatic and renal cytochrome (CYP) P450-mediated arachidonic acid metabolism. 2. Our results demonstrated that acute As(III) treatment (12.5 mg/kg) altered CYP epoxygenases, CYP ω-hydroxylases and EPHX2 mRNA levels that were isozyme and tissue specific. 3. Furthermore, As(III) increased the formation of epoxyeicosatrienoic acids (EETs) in the kidney without affecting their levels in the lung or liver. In addition, acute As(III) treatment increased dihydroxyeicosatrienoic acid (DHETs) formation in the lung, while it did not affect liver DHETs formation and decreased kidney DHETs formation. 4. As(III) also increased total epoxygenases activity in the lung while it decreased its levels in the kidney and had no effect on the liver. Furthermore, As(III) increased 20-hydroxyeicosatetraenoic acid formation in the liver while it decreased its formation in the kidney. 5. Lastly, As(III) increased soluble epoxide hydrolase activity in the lung, while it decreased its levels in the kidney and had no effect on the liver. In conclusion, this is the first demonstration that As(III) alters arachidonic acid metabolism in a tissue specific manner.

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.