Preclinical and clinical evidence for the role of resveratrol in the treatment of cardiovascular diseases.

Cardiovascular disease is the leading cause of death worldwide. Despite advancements in diagnosis and treatment of cardiovascular disease, the incidence of cardiovascular disease is still rising. Therefore, new lines of medications are needed to treat the growing population of patients with cardiovascular disease. Although the majority of the existing pharmacotherapies for cardiovascular disease are synthesized molecules, natural compounds, such as resveratrol, are also being tested. Resveratrol is a non-flavonoid polyphenolic compound, which has several biological effects. Preclinical studies have provided convincing evidence that resveratrol has beneficial effects in animal models of hypertension, atherosclerosis, stroke, ischemic heart disease, arrhythmia, chemotherapy-induced cardiotoxicity, diabetic cardiomyopathy, and heart failure. Although not fully delineated, some of the beneficial cardiovascular effects of resveratrol are mediated through activation of silent information regulator 1 (SIRT1), AMP-activated protein kinase (AMPK), and endogenous anti-oxidant enzymes. In addition to these pathways, the anti-inflammatory, anti-platelet, insulin-sensitizing, and lipid-lowering properties of resveratrol contribute to its beneficial cardiovascular effects. Despite the promise of resveratrol as a treatment for numerous cardiovascular diseases, the clinical studies for resveratrol are still limited. In addition, several conflicting results from trials have been reported, which demonstrates the challenges that face the translation of the exciting preclinical findings to humans. Herein, we will review much of the preclinical and clinical evidence for the role of resveratrol in the treatment of cardiovascular disease and provide information about the physiological and molecular signaling mechanisms involved. This article is part of a Special Issue entitled: Resveratrol: Challenges in translating pre-clinical findings to improved patient outcomes.

AMPK-dependent inhibitory phosphorylation of ACC is not essential for maintaining myocardial fatty acid oxidation.

RATIONALE: The energy sensor AMP-activated protein kinases (AMPK) is thought to play an important role in regulating myocardial fatty acid oxidation (FAO) via its phosphorylation and inactivation of acetyl coenzyme A carboxylase (ACC). However, studies supporting this have not directly assessed whether the maintenance of FAO rates and subsequent cardiac function requires AMPK-dependent inhibitory phosphorylation of ACC. OBJECTIVE: To determine whether preventing AMPK-mediated inactivation of ACC influences myocardial FAO or function. METHODS AND RESULTS: A double knock-in (DKI) mouse (ACC-DKI) model was generated in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser221 (Ser212 mouse) on ACC2 were mutated to prevent AMPK-dependent inhibitory phosphorylation of ACC. Hearts from ACC-DKI mice displayed a complete loss of ACC phosphorylation at the AMPK phosphorylation sites. Despite the inability of AMPK to regulate ACC activity, hearts from ACC-DKI mice displayed normal basal AMPK activation and cardiac function at both standard and elevated workloads. In agreement with the inability of AMPK in hearts from ACC-DKI mice to phosphorylate and inhibit ACC, there was a significant increase in cardiac malonyl-CoA content compared with wild-type mice. However, cardiac FAO rates were comparable between wild-type and ACC-DKI mice at baseline, during elevated workloads, and after a more stressful condition of myocardial ischemia that is known to robustly activate AMPK. CONCLUSIONS: Our findings show AMPK-dependent inactivation of ACC is not essential for the control of myocardial FAO and subsequent cardiac function during a variety of conditions involving AMPK-independent and AMPK-dependent metabolic adaptations.

The anti-proliferative effect of metformin in triple-negative MDA-MB-231 breast cancer cells is highly dependent on glucose concentration: implications for cancer therapy and prevention.

BACKGROUND: Metformin has been shown to have a strong anti-proliferative effect in many breast cancer cell lines, mainly due to the activation of the energy sensing kinase, AMP-activated protein kinase (AMPK). MDA-MB-231 cells are aggressive and invasive breast cancer cells that are known to be resistant to several anti-cancer agents as well as to the anti-proliferative effect of metformin. As metformin is a glucose lowering drug, we hypothesized that normoglycemia will sensitize MDA-MB-231 cells to the anti-proliferative effect of metformin. METHODS: MDA-MB-231 cells were treated with increasing metformin concentrations in hyperglycemic or normoglycemic conditions. The growth inhibitory effect of metformin was assessed by MTT assay. The expression of several proteins involved in cell proliferation was measured by Western blotting. RESULTS: In agreement with previous studies, treatment with metformin did not inhibit the growth of MDA-MB-231 cells cultured in hyperglycemic conditions. However, metformin significantly inhibited MDA-MB-231 growth when the cells were cultured in normoglycemic conditions. In addition, we show that metformin-treatment of MDA-MB-231 cells cultured in normoglycemic conditions and not in hyperglycemic conditions caused a striking activation of AMPK, and an AMPK-dependent inhibition of multiple molecular signaling pathways known to control protein synthesis and cell proliferation. CONCLUSION: Our data show that normoglycemia sensitizes the triple negative MDA-MB-231 breast cancer cells to the anti-proliferative effect of metformin through an AMPK-dependent mechanism. GENERAL SIGNIFICANCE: These findings suggest that tight normoglycemic control may enhance the anti-proliferative effect of metformin in diabetic cancer patients.

Role of cytochrome P450-mediated arachidonic acid metabolites in the pathogenesis of cardiac hypertrophy.

A plethora of studies have demonstrated the expression of cytochrome P450 (CYP) and soluble epoxide hydrolase (sEH) enzymes in the heart and other cardiovascular tissues. In addition, the expression of these enzymes is altered during several cardiovascular diseases (CVDs), including cardiac hypertrophy (CH). The alteration in CYP and sEH expression results in derailed CYP-mediated arachidonic acid (AA) metabolism. In animal models of CH, it has been reported that there is an increase in 20-hydroxyeicosatetraenoic acid (20-HETE) and a decrease in epoxyeicosatrienoic acids (EETs). Further, inhibiting 20-HETE production by CYP ω-hydroxylase inhibitors and increasing EET stability by sEH inhibitors have been proven to protect against CH as well as other CVDs. Therefore, CYP-mediated AA metabolites 20-HETE and EETs are potential key players in the pathogenesis of CH. Some studies have investigated the molecular mechanisms by which these metabolites mediate their effects on cardiomyocytes and vasculature leading to pathological CH. Activation of several intracellular signaling cascades, such as nuclear factor of activated T cells, nuclear factor kappa B, mitogen-activated protein kinases, Rho-kinases, Gp130/signal transducer and activator of transcription, extracellular matrix degradation, apoptotic cascades, inflammatory cytokines, and oxidative stress, has been linked to the pathogenesis of CH. In this review, we discuss how 20-HETE and EETs can affect these signaling pathways to result in, or protect from, CH, respectively. However, further understanding of these metabolites and their effects on intracellular cascades will be required to assess their potential translation to therapeutic approaches for the prevention and/or treatment of CH and heart failure.

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.

Differential effects of soluble epoxide hydrolase inhibition and CYP2J2 overexpression on postischemic cardiac function in aged mice.

Cardioprotective effects of epoxyeicosatrienoic acids (EETs) have been demonstrated in models of young mice with either the cardiomyocyte specific over-expression of cytochrome P450 2J2 (CYP2J2 Tr) or deletion of soluble epoxide hydrolase (sEH null). In this study we examined differences in EET-induced cardioprotection in young (2 months) and aged (12 months) CYP2J2 Tr and sEHnull mice using Langendorff isolated perfused heart model. Improved postischemic functional recovery was observed in both young and aged sEH null mice compared to age matched WT. Conversely, the cardioprotective effect observed in young CYP2J2 Tr was lost in aged CYP2J2 Tr mice. The loss of cardioprotection in aged CYP2J2 Tr was regained following perfusion with the sEH inhibitor t-AUCB. Data demonstrated increased levels of leukotoxin diol (DiHOME) and oxidative stress as well decreased protein phosphatase 2A (PP2A) activation in aged CYP2J2 Tr. In conclusion, inhibition of sEH and EET-induced cardioprotection is maintained in aged mice. However, the loss of protective effects observed in aged CYP2J2 Tr might be attributed to increased levels of DiHOME, oxidative stress and/or decreased PP2A activity.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: Male Sprague-Dawley rats were treated with TUPS (0.65 mg kg(-1) day(-1), p.o.), isoprenaline (5 mg kg(-1) day(-1), i.p.) or the combination of both. In vitro H9c2 cells were treated with isoprenaline (100 µM) in the presence and absence of either TUPS (1 µM) or 11,12 EET (1 µM). The expression of hypertrophic, fibrotic markers and different CYP genes were determined by real-time PCR. KEY RESULTS: Isoprenaline significantly induced the hypertrophic, fibrotic markers as well as the heart to body weight ratio, which was significantly reversed by TUPS. Isoprenaline also caused an induction of CYP1A1, CYP1B1, CYP2B1, CYP2B2, CYP4A3 and CYP4F4 gene expression and TUPS significantly inhibited this isoprenaline-mediated effect. Moreover, isoprenaline significantly reduced 5,6-, 8,9-, 11,12- and 14,15-EET and increased their corresponding 8,9-, 11,12- and 14,15-dihydroxyeicosatrienoic acid (DHET) and the 20-HETE metabolites. TUPS abolished these isoprenaline-mediated changes in arachidonic acid (AA) metabolites. In H9c2 cells, isoprenaline caused a significant induction of ANP, BNP and EPHX2 mRNA levels. Both TUPS and 11,12-EET significantly decreased this isoprenaline-mediated induction of ANP, BNP and EPHX2. CONCLUSIONS AND IMPLICATIONS: TUPS partially protects against isoprenaline-induced cardiac hypertrophy, which confirms the role of sEH and CYP enzymes in the development of cardiac hypertrophy.

Determination of the dominant arachidonic acid cytochrome p450 monooxygenases in rat heart, lung, kidney, and liver: protein expression and metabolite kinetics.

Cytochrome P450 (P450)-derived arachidonic acid (AA) metabolites serve pivotal physiological roles. Therefore, it is important to determine the dominant P450 AA monooxygenases in different organs. We investigated the P450 AA monooxygenases protein expression as well as regioselectivity, immunoinhibition, and kinetic profile of AA epoxygenation and hydroxylation in rat heart, lung, kidney, and liver. Thereafter, the predominant P450 epoxygenases and P450 hydroxylases in these organs were characterized. Microsomes from heart, lung, kidney, and liver were incubated with AA. The protein expression of CYP2B1/2, CYP2C11, CYP2C23, CYP2J3, CYP4A1/2/3, and CYP4Fs in the heart, lung, kidney, and liver were determined by Western blot analysis. The levels of AA metabolites were determined by liquid chromatography-electrospray ionization mass spectroscopy. This was followed by determination of regioselectivity, immunoinhibition effect, and the kinetic profile of AA metabolism. AA was metabolized to epoxyeicosatrienoic acids and 19- and 20-hydroxyeicosatetraenoic acid in the heart, lung, kidney, and liver but with varying metabolic activities and regioselectivity. Anti-P450 antibodies were found to differentially inhibit AA epoxygenation and hydroxylation in these organs. Our data suggest that the predominant epoxygenases are CYP2C11, CYP2B1, CYP2C23, and CYP2C11/CYP2C23 for the heart, lung, kidney, and liver, respectively. On the other hand, CYP4A1 is the major ω-hydroxylase in the heart and kidney; whereas CYP4A2 and/or CYP4F1/4 are probably the major hydroxlases in the lung and liver. These results provide important insights into the activities of P450 epoxygenases and P450 hydroxylases-mediated AA metabolism in different organs and their associated P450 protein levels.

Chronic doxorubicin cardiotoxicity modulates cardiac cytochrome P450-mediated arachidonic acid metabolism in rats.

Doxorubicin [(DOX) Adriamycin] is an effective anticancer agent whose major limiting side effect is cardiotoxicity. This cardiotoxicity is predicted only by the cumulative dose of DOX where the clinical situation involves chronic drug administration. Therefore, we investigate the effect of chronic DOX cardiotoxicity on expression of the cardiac cytochrome P450 (P450) enzymes and arachidonic acid (AA) metabolism in male Sprague-Dawley (SD) rats. The chronic toxicity was induced by multiple intraperitoneal injections for a cumulative dose of 15 mg/kg divided into six injections within 2 weeks. After 14 days of the last injection, the heart, liver, and kidney were harvested, and the expression of different genes was determined by real-time polymerase chain reaction. In addition, microsomal protein from the heart was prepared and incubated with AA. Thereafter, different AA metabolites were analyzed by liquid chromatography-electrospray ionization-mass spectrometry. The chronic DOX cardiotoxicity significantly induced gene expression of hypertrophic markers, apoptotic markers, CYP2E1, CYP4A3, CYP4F1, CYP4F5, and soluble epoxide hydrolase (sEH) enzyme, which was accompanied by an increase in the activity of P450 ω-hydroxylases and sEH. In addition, both the sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid, and the ω-hydroxylase inhibitor, N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016), significantly prevented the DOX-mediated induction of the hypertrophic markers in the cardiac-derived H9c2 cells, which further confirms the role of these enzymes in DOX cardiotoxicity. Furthermore, gene expression of P450 and sEH was altered in an organ-specific manner. As a result, the chronic DOX administration leads to an imbalance between P450-mediated cardiotoxic and cardioprotective pathways. Therefore, P450 ω-hydroxylases and sEH might be considered as novel targets to prevent and/or treat DOX cardiotoxicity.

Acute arsenic toxicity alters cytochrome P450 and soluble epoxide hydrolase and their associated arachidonic acid metabolism in C57Bl/6 mouse heart.

Acute arsenic (As(III)) exposure has been reported to cause cardiac toxicity, however this toxicity was never linked to the disturbance in cytochrome P450 (P450)-mediated arachidonic acid metabolism. Therefore, we investigated the effect of acute As(III) toxicity on the expression of P450 and soluble epoxide hydrolase (sEH) and their associated arachidonic acid metabolism in mice hearts. As(III) toxicity was induced by a single intraperitoneal injection of 12.5 mg/kg of As(III). Our results showed that As(III) treatment caused a significant induction of the cardiac hypertrophic markers in addition to Cyp1b1, Cyp2b, Cyp2c, Cyp4f, and sEH gene expression in mice hearts. Furthermore, As(III) increased sEH protein expression and activity in hearts with a consequent decrease in 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) formation. Whereas the formation of 8,9-, 11,12-, 14,15-dihydroxyeicosatrienoic acids (DHETs) was significantly increased. As(III) also increased sEH mRNA and protein expression levels in addition to the hypertrophic markers which was reversed by knockdown of sEH in H9c2 cells. In conclusion, acute As(III) toxicity alters the expression of several P450s and sEH enzymes with a consequent decrease in the cardioprotective EETs which may represent a novel mechanism by which As(III) causes progressive cardiotoxicity. Furthermore, inhibiting sEH might represent a novel therapeutic approach to prevent As(III)-induced hypertrophy.

Acute doxorubicin toxicity differentially alters cytochrome P450 expression and arachidonic acid metabolism in rat kidney and liver.

The use of doxorubicin (DOX) is limited by significant cardiotoxicity, nephrotoxicity, and hepatotoxicity. We have previously shown that DOX cardiotoxicity induces several cardiac cytochrome P450 (P450) enzymes with subsequent alteration in P450-mediated arachidonic acid metabolism. Therefore, in the current study, we investigated the effect of acute DOX toxicity on P450 expression and arachidonic acid metabolism in the kidney and liver of male Sprague-Dawley rats. Acute DOX toxicity was induced by a single intraperitoneal injection (15 mg/kg) of the drug. After 6 and 24 h, the kidneys and livers were harvested, and several P450 gene and protein expressions were determined by real-time polymerase chain reaction and Western blot analyses, respectively. Kidney and liver microsomal protein from control or DOX-treated rats was incubated with arachidonic acid, and its metabolites were determined by liquid chromatography-electron spray ionization-mass spectrometry. Our results showed that acute DOX toxicity caused an induction of CYP1B1 and CYP4A enzymes and an inhibition of CYP2B1 and CYP2C11 in both the kidney and liver. CYP2E1 was induced and soluble epoxide hydrolase (sEH) was inhibited in the kidney only. In addition, DOX toxicity caused a significant increase in epoxyeicosatrienoic acids formation in the kidney and a significant increase in 20-hydroxyeicosatetraenoic acid formation in both the kidney and the liver. In conclusion, acute DOX toxicity alters the expression of several P450 and sEH enzymes in an organ-specific manner. These changes can be attributed to DOX-induced inflammation and resulted in altered P450-mediated arachidonic acid metabolism.

Inhibition of soluble epoxide hydrolase confers cardioprotection and prevents cardiac cytochrome P450 induction by benzo(a)pyrene.

We recently demonstrated that benzo(a)pyrene (BaP) causes cardiac hypertrophy by altering arachidonic acid metabolism through the induction of the expression of CYP ω-hydroxylases and soluble epoxide hydrolase (sEH) enzymes. The inhibition of CYP ω-hydroxylase enzymes partially reversed the BaP-induced cardiac hypertrophy. Therefore, it is important to examine whether the inhibition of sEH also confers cardioprotection. For this purpose, male Sprague-Dawley rats were injected intraperitoneally daily with either the sEH inhibitor 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS; 0.65 mg/kg), BaP (20 mg/kg), or the combination of BaP (20 mg/kg) and TUPS (0.65 mg/kg) for 7 days. Thereafter, the heart, liver, and kidney were harvested, and the heart to body weight ratio was measured. The expression of the hypertrophic markers, sEH, heme oxygenase-1, and CYP450 enzymes was determined. Our results demonstrate that BaP alone significantly induced the expression of sEH and CYP ω-hydroxylases in the heart, liver, and kidney tissues. Treatment with TUPS significantly reversed the BaP-mediated induction of the hypertrophic markers, completely prevented the increase in the heart to body weight ratio, and reduced the BaP-induced CYP1A1, CYP1B1, CYP4F4, and CYP4F5 genes in the heart. The current study demonstrates the cardioprotective effect of sEH inhibitor, TUPS, against BaP-induced cardiac hypertrophy and further confirms the role of sEH and CYP450 enzymes in the development of cardiac hypertrophy.

Alteration of cardiac cytochrome P450-mediated arachidonic acid metabolism in response to lipopolysaccharide-induced acute systemic inflammation.

Cytochrome P450 (CYP) generated cardioprotective metabolites, epoxyeicosatrienoic acids (EETs), and cardiotoxic metabolites, hydroxyeicosatetraenoic acids (HETEs) levels are determined by many factors, including the induction or repression of the CYP enzymes, responsible for their formation. Therefore, we examined the effect of acute inflammation on the expression of CYP epoxygenases and CYP omega-hydroxylases in the heart, kidney, and liver and the cardiac CYP-mediated arachidonic acid metabolism. For this purpose, male Sprague-Dawley rats were injected intraperitoneally with LPS (1mg/kg). After 6, 12, or 24h, the tissues were harvested and the expression of CYP genes and protein levels were determined using real time-PCR, and Western blot analyses, respectively. Arachidonic acid metabolites formations were determined by liquid chromatography-electron spray ionization-mass spectrometry LC-ESI-MS. Our results showed that inflammation significantly decreased the CYP epoxygenases expression in the heart, kidney and liver with a concomitant decrease in the EETs produced by these enzymes. In contrast to CYP expoxygenses, inflammation differentially altered CYP omega-hydroxylases expression with a significant increase in 20-HETE formation. The present study demonstrates for the first time that acute inflammation decreases CYP epoxygenases and their associated cardioprotective metabolites, EETs while on the other hand increases CYP omega-hydroxylases and their associated cardiotoxic metabolites, 20-HETE. These changes may be involved in the development and/or progression of cardiovascular diseases by inflammation.

Effect of cytochrome P450 polymorphism on arachidonic acid metabolism and their impact on cardiovascular diseases.

Cardiovascular diseases (CVDs) remain the leading cause of death in the developed countries. Taking into account the mounting evidence about the role of cytochrome P450 (CYP) enzymes in cardiovascular physiology, CYP polymorphisms can be considered one of the major determinants of individual susceptibility to CVDs. One of the important physiological roles of CYP enzymes is the metabolism of arachidonic acid. CYP epoxygenases such as CYP1A2, CYP2C, and CYP2J2 metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which generally possess vasodilating, anti-inflammatory, anti-apoptotic, anti-thrombotic, natriuretic, and cardioprotective effects. Therefore, genetic polymorphisms causing lower activity of these enzymes are generally associated with an increased risk of several CVDs such as hypertension and coronary artery disease. EETs are further metabolized by soluble epoxide hydrolase (sEH) to the less biologically active dihydroxyeicosatrienoic acids (DHETs). Therefore, sEH polymorphism has also been shown to affect arachidonic acid metabolism and to be associated with CVDs. On the other hand, CYP omega-hydroxylases such as CYP4A11 and CYP4F2 metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) which has both vasoconstricting and natriuretic effects. Genetic polymorphisms causing lower activity of these enzymes are generally associated with higher risk of hypertension. Nevertheless, some studies have denied the association between polymorphisms in the arachidonic acid pathway and CVDs. Therefore, more research is needed to confirm this association and to better understand the pathophysiologic mechanisms behind it.

2,3,7,8-Tetrachlorodibenzo-p-dioxin and beta-naphthoflavone induce cellular hypertrophy in H9c2 cells by an aryl hydrocarbon receptor-dependant mechanism.

Cigarette smoke is a major risk factor for cardiovascular diseases. It contains thousands of compounds that activate the aryl hydrocarbon receptor (AhR). In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent AhR ligand, has been shown to cause cardiotoxic effects in several in vivo models. Although induction of CYP1 family is the most important effect of AhR activation, the role of CYP1 induction in mediating the cardiotoxic effect of TCDD is usually overlooked. Therefore, we investigated whether AhR activation causes a hypertrophic effect in H9c2 cells and we related this effect to changes in CYP gene expression. In the current study, the cardiac derived H9c2 cells were treated with two AhR ligands, TCDD and beta-naphthoflavone (BNF), for 24 and 48h. The expression of the hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and several CYP genes were measured by real-time PCR. Treatment of H9c2 cells with TCDD or BNF for 24h caused a significant induction of CYP1A1, CYP1B1, and CYP4A1; however, there was no change in the expression of other genes. On the other hand, treatment of the cells with TCDD or BNF for 48h caused a significant induction of the hypertrophic markers, ANP and BNP, and several CYP genes such as CYP1A1, CYP1B1, CYP2E1, CYP2J3, and CYP4F4 parallel to a significant increase in the cell surface area. Neither TCDD nor BNF increased the oxidative stress in H9c2 cells at all concentrations tested. Interestingly, resveratrol, an AhR antagonist, protected the cells from TCDD-induced hypertrophy. In conclusion, AhR ligands caused a hypertrophic effect in H9c2 cells which was associated with induction of several CYP genes which can be prevented by resveratrol.

Acute doxorubicin cardiotoxicity alters cardiac cytochrome P450 expression and arachidonic acid metabolism in rats.

Doxorubicin (DOX) is a potent anti-neoplastic antibiotic used to treat a variety of malignancies; however, its use is limited by dose-dependent cardiotoxicity. Moreover, there is a strong correlation between cytochrome P450 (CYP)-mediated arachidonic acid metabolites and the pathogenesis of many cardiovascular diseases. Therefore, in the current study, we have investigated the effect of acute DOX toxicity on the expression of several CYP enzymes and their associated arachidonic acid metabolites in the heart of male Sprague-Dawley rats. Acute DOX toxicity was induced by a single intraperitoneal injection of 15 mg/kg of the drug. Our results showed that DOX treatment for 24 h caused a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A1, CYP4A3, CYP4F1, CYP4F4, and EPHX2 gene expression in the heart of DOX-treated rats as compared to the control. Similarly, there was a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A, and sEH proteins after 24 h of DOX administration. In the heart microsomes, acute DOX toxicity significantly increased the formation of 20-HETE which is consistent with the induction of the major CYP omega-hydroxylases: CYP4A1, CYP4A3, CYP4F1, and CYP4F4. On the other hand, the formation of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) was significantly reduced, whereas the formation of their corresponding dihydroxyeicosatrienoic acids was significantly increased. The decrease in the cardioprotective EETs can be attributed to the increase of sEH activity parallel to the induction of the EPHX2 gene expression in the heart of DOX-treated rats. In conclusion, acute DOX toxicity alters the expression of several CYP and sEH enzymes with a consequent alteration in arachidonic acid metabolism. These results may represent a novel mechanism by which this drug causes progressive cardiotoxicity.

3-methylcholanthrene and benzo(a)pyrene modulate cardiac cytochrome P450 gene expression and arachidonic acid metabolism in male Sprague Dawley rats.

BACKGROUND AND PURPOSE: There is a strong correlation between cytochrome P450 (P450)-dependent arachidonic acid metabolism and the pathogenesis of cardiac hypertrophy. Several aryl hydrocarbon receptor (AhR) ligands were found to alter P450-dependent arachidonic acid metabolism. Here, we have investigated the effect of 3-methylcholanthrene (3-MC) and benzo(a)pyrene (BaP), two AhR ligands, on the development of cardiac hypertrophy. EXPERIMENTAL APPROACH: Male Sprague Dawley rats were injected (i.p.) daily with either 3-MC (10 mg kg(-1)) or BaP (20 mg kg(-1)) for 7 days. Then hearts were removed, and the heart to body weight ratio and the gene expression of the hypertrophic markers and P450 genes were determined. Levels of arachidonic acid metabolites were determined by liquid chromatography-electron spray ionization-mass spectrometry. KEY RESULTS: Both 3-MC and BaP increased the heart to body weight ratio as well as the hypertrophic markers, atrial natriuretic peptide and brain natriuretic peptide. 3-MC and BaP treatment increased the gene expression of CYP1A1, CYP1B1, CYP2E1, CYP4F4, CYP4F5 and soluble epoxide hydrolase. Both 3-MC and BaP treatments increased the dihydroxyeicosatrienoic acids (DHETs) : epoxyeicosatrienoic acids (EETs) ratio and the 20-hydroxyeicosatetraenoic acid (20-HETE) : total EETs ratio. Treatment with benzo(e)pyrene, an isomer of BaP that is a poor ligand for the AhR, did not induce cardiac hypertrophy in rats, confirming the role of AhR in the development of cardiac hypertrophy. Treatment with the omega-hydroxylase inhibitor, HET0016, significantly reversed BaP-induced cardiac hypertrophy. CONCLUSIONS AND IMPLICATIONS: 3-MC and BaP induce cardiac hypertrophy by increasing the ratio of DHETs : EETs and/or the ratio of 20-HETE : total EETs, through increasing soluble epoxide hydrolase activity.

Role of NF-kappaB in the regulation of cytochrome P450 enzymes.

Nuclear factor kappa B (NF-kappaB) is an important transcription factor that regulates a wide spectrum of genes including cytochrome P450 (CYP), the most important family of drug metabolizing enzymes. Therefore, in this review, we addressed the potential role of NF-kappaB in CYP regulation. We proposed three mechanisms by which NF-kappaB can regulate CYP expression and activity. First, NF-kappaB can directly regulate the expression of CYP1A1, CYP2B1/2, CYP2C11, CYP2D5, CYP2E1, CYP3A7, and CYP27B1 through binding to the promoter region of these genes. Second, NF-kappaB indirectly regulates the transcription of CYP genes through mutual repression with some nuclear receptors that are involved in CYP regulation such as AhR, CAR, GR, PXR, RXR, PPAR, FXR, and LXR. Finally, NF-kappaB can regulate CYP activity at post-transcriptional level by inducing heme oxygenase or by affecting the CYP protein stability. In addition, increased inflammatory mediators, oxidative stress, and subsequent NF-kappaB activation have been demonstrated in many conditions such as inflammatory bowel diseases, rheumatoid arthritis, psychological stress, diabetes, aging, cancer, renal diseases, and congestive heart failure. Meanwhile, there is a significant alteration of CYP expression and activity in these diseases. Therefore, we propose that NF-kappaB could be one of the links between inflammation, oxidative stress, and CYP regulation in these diseases. In conclusion, NF-kappaB plays a crucial role in the regulation of CYP through several mechanisms and this role can explain the altered CYP regulation in many conditions.

Induction of several cytochrome P450 genes by doxorubicin in H9c2 cells.

Doxorubicin is a potent anti-neoplastic antibiotic used to treat a wide variety of malignancies; however, its use is limited by dose dependent cardiotoxicity. There is indirect evidence suggesting that doxorubicin cardiotoxicity is CYP-mediated. In the current study, we investigated the effect of doxorubicin on hypertrophic markers, and different CYP gene expression in cardiac derived H9c2 cells. H9c2 cells were incubated with increasing concentrations of doxorubicin and the expressions of different genes were determined by real-time PCR. Our results demonstrate that multiple CYP genes are expressed in H9c2 cells and the level of expression from the highest to the lowest were; CYP1B1, CYP2B1, CYP2J3, CYP1A1, CYP2C11, CYP2C23, CYP2E1, CYP1A2, and CYP2B2. Doxorubicin treatment caused an induction of the hypertrophic markers, ANP and BNP. In addition, doxorubicin caused a significant induction of CYP1A1, CYP1A2, CYP1B1, CYP2B2, CYP2E1, and CYP2J3 gene expression in a concentration-dependent manner. However, only the highest concentration tested, 10 muM, caused an induction of CYP2C11; whereas, CYP2B1 and CYP2C23 were not altered. Our findings demonstrate that doxorubicin induces the hypertrophic markers, ANP and BNP as well as several CYP genes in H9c2 cells. Doxorubicin-mediated CYP induction may represent a novel mechanism by which this drug induces cardiotoxicity.

Modulation of cytochrome P450 gene expression and arachidonic acid metabolism during isoproterenol-induced cardiac hypertrophy in rats.

Several cytochrome P450 (P450) enzymes have been identified in the heart, and their levels have been reported to be altered during cardiac hypertrophy. Moreover, there is a strong correlation between P450-mediated arachidonic acid metabolites and the pathogenesis of cardiac hypertrophy. Therefore, we investigated the effect of isoproterenol-induced cardiac hypertrophy on the expression of several P450 genes and their associated P450-derived metabolites of arachidonic acid. Cardiac hypertrophy was induced by seven daily i.p. injections of 5 mg/kg isoproterenol. Thereafter, the heart, lung, liver, and kidney were harvested, and the expression of different genes was determined by real-time polymerase chain reaction. Heart microsomal protein from control or isoproterenol treated rats was incubated with 50 microM arachidonic acid, and arachidonic acid metabolites were determined by liquid chromatography-electron spray ionization-mass spectrometry. Our results show that isoproterenol treatment significantly increased the heart/body weight ratio and the hypertrophic markers. In addition, there was a significant induction of CYP1A1, CYP1B1, CYP4A3, and soluble epoxide hydrolase and a significant inhibition of CYP2C11 and CYP2E1 in the hypertrophied hearts as compared with the control. CYP1A1, CYP2E1, and CYP4A3 gene expression was induced in the kidney, and CYP4A3 was induced in the liver of isoproterenol-treated rats. Isoproterenol treatment significantly reduced 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid formation and significantly increased their corresponding 8,9-, and 14,15-dihydroxyeicosatrienoic acid and the 20-hydroxyeicosatetraenoic acid metabolite. In conclusion, isoproterenol-induced cardiac hypertrophy alters arachidonic acid metabolism and its associated P450 enzymes, suggesting their role in the development and/or progression of cardiac hypertrophy.