Inflammatory mediators, oxidative stress and genetic disturbance in rheumatoid arthritis rats supported by alfalfa seeds metabolomic constituents via blocking interleukin-1receptor.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by aggressive cartilage and bone erosion. This work aimed to evaluate the metabolomic profile of Medicago sativa L. (MS) (alfalfa) seeds and explore its therapeutic impact against RA in rats. Arthritis was induced by complete Freund's adjuvant (CFA) and its severity was assessed by the arthritis index. Treatment with MS seeds butanol fraction and interlukin-1 receptor antagonist (IL-1RA) were evaluated through measuring interlukin-1 receptor (IL-1R) type 1 gene expression, interlukin-1 beta (IL-1ß), oxidative stress markers, C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), caspase-3 (Cas-3), intracellular adhesion molecule-1 (ICAM-1), DNA fragmentation, and chromosomal damage. Total phenolics/ flavonoids content in the ethyl acetate, butanol fraction and crude extract of MS seeds were estimated. The major identified compounds were Quercetin, Trans-taxifolin, Gallic acid, 7,4'-Dihydroxyflavone, Cinnamic acid, Kudzusaponin SA4, Isorhamnetin 3-O-beta-D-2'',3'',4''-triacetylglucopyranoside, Apigenin, 5,7,4'-Trihydroxy-3'-methoxyflavone, Desmethylxanthohumol, Pantothenic acid, Soyasapogenol E, Malvidin, Helilandin B, Stigmasterol, and Wairol. Treatment with MS seeds butanol fraction and IL-1RA enhanced all the biochemical parameters and the histopathological features of the ankle joint. In conclusion, Trans-taxifolin was isolated for the first time from the genus Medicago. MS butanol fraction seeds extract and IL-1 RA were considered as anti-rheumatic agents.

HPLC and GC-MS-based metabolic profiles and in vivo anticonvulsant, sedative, and antinociceptive potentials of truffles Tirmania nivea and Tirmania pinoyi hydromethanolic extracts in mice.

GC-MS and HPLC analyses of the hydromethanolic extracts of the truffles Tirmania nivea (TN) and Tirmania pinoyi (TP) revealed the presence of 18 metabolites and 11 polyphenols, respectively. In vivo, TP extract protected against subcutaneous pentylenetetrazole (scPTZ) and maximal electric shock (MES)-induced convulsions faster than TN extract. TP extract (100 and 300 mg/kg) showed 100% protection and longer duration than TN extract in the scPTZ test. Similarly, at 300 mg/kg, TP demonstrated a quicker start (75%) and longer duration of action (100%) than TN in MES test. In the scPTZ test, ED50 of TP demonstrated greater anticonvulsant efficacy than that of TN. In mice given TP and TN treatments, the brain GABA levels noticeably increased. TP (100 and 300 mg/kg) produced a notable sedative effect in open-field test, whereas TN (100 or 300 mg/kg) and TP (300 mg/kg) reduced sleep latency by 52%, 45%, and 79%, respectively. In writhing test, TN (100 or 300 mg/kg) significantly enhanced analgesic efficacy by 50 and 87%, respectively. Comparatively, in formalin test, TP and TN at a dosage of 300 mg/kg decreased the length of the licking by 34 and 59%, respectively. For the first time, this study explains the anticonvulsant, sedative, central, and peripheral analgesic activities of truffle extracts.

Bioactive compounds and therapeutic role of Brassica oleracea L. seeds in rheumatoid arthritis rats via regulating inflammatory signalling pathways and antagonizing interleukin-1 receptor action.

CONTEXT: Rheumatoid arthritis (RA) is a chronic, progressive autoimmune disease characterized by aggressive and systematic polyarthritis. OBJECTIVE: The present study aimed to isolate and identify the phenolic constituents in Brassica oleracea L. (Brassicaceae) seeds methanolic extract and evaluates its effect against rheumatoid arthritis in rats referring to the new therapy; interleukin-1 receptor antagonist (IL-1RA). MATERIALS AND METHODS: The GC/MS profiling of the plant was determined. Arthritis induction was done using complete Freund's adjuvant. Arthritis severity was assessed by percentage of edema and arthritis index. IL-1 receptor type I gene expression, interleukin-1ß (IL-1ß), oxidative stress markers, protein content, inflammatory mediators, prostaglandin-E2 (PGE2), genetic abnormalities and the histopathological features of ankle joint were evaluated. RESULTS: For the first time twelve phenolic compounds had been isolated from the seeds extract. Treatment with extract and IL-1RA improved the tested parameters by variable degrees. CONCLUSIONS: RA is an irreversible disease, where its severity increases with the time of induction. Brassica oleracea L. seeds extract is considered as a promising anti-arthritis agent. IL-1 RA may be considered as an unusual therapeutic agent for RA disease. More studies are needed to consider the seeds extract as a nutraceutical agent and to recommend IL-1RA as a new RA drug.

Discovery of 4-benzyloxy and 4-(2-phenylethoxy) chalcone fibrate hybrids as novel PPARα agonists with anti-hyperlipidemic and antioxidant activities: Design, synthesis and in vitro/in vivo biological evaluation.

In the current work, a series of novel 4-benzyloxy and 4-(2-phenylethoxy) chalcone fibrate hybrids (10a-o) and (11a-e) were synthesized and evaluated as new PPARα agonists in order to find new agents with higher activity and fewer side effects. The 2-propanoic acid derivative 10a and the 2-butanoic acid congener 10i showed the best overall PPARα agonistic activity showing Emax% values of 50.80 and 90.55%, respectively, and EC50 values of 8.9 and 25.0 µM, respectively, compared to fenofibric acid with Emax = 100% and EC50 = 23.22 µM, respectively. These two compounds also stimulated carnitine palmitoyltransferase 1A gene transcription in HepG2 cells and PPARα protein expression. Molecular docking simulations were performed for the newly synthesized compounds to study their predicted binding pattern and energies in PPARα active site to rationalize their promising activity. In vivo, compounds 10a and 10i elicited a significant hypolipidemic activity improving the lipid profile in triton WR-1339-induced hyperlipidemic rats, including serum triglycerides, total cholesterol, LDL, HDL and VLDL levels. Compound 10i possessed better anti-hyperlipidemic activity than 10a. At a dose of 200 mg/kg, it demonstrated significantly lower TC, TG, LDL and VLDL levels than that of fenofibrate at the same dose with similar HDL levels. Compounds 10i and 10a possessed atherogenic indices (CRR, AC, AI, CRI-II) like that of fenofibrate. Additionally, a promising antioxidant activity indicated by the increased tissue reduced glutathione and plasma total antioxidant capacity with decreased plasma malondialdehyde levels was demonstrated by compounds 10a and 10i. No histopathological alterations were recorded in the hepatic tissue of compound 10i (200 mg/kg).

HPLC-ESI/MS profiling, phytoconstituent isolation and evaluation of renal function, oxidative stress and inflammation in gentamicin-induced nephrotoxicity in rats of Ficus spragueana Mildbr. & Burret.

Ficus spragueana Mildbr. & Burret (family Moraceae) was reported to have various biological activities. However, its activity in treatment of renal injury has not been investigated yet. The current study aimed to evaluate the effects of F. spragueana leaf extract on nephrotoxicity caused by gentamicin. Gentamicin is an important broad-spectrum antibiotic; nevertheless, it exhibits serious nephrotoxic adverse effects. HPLC-ESI/MS spectrometric analysis of the extract revealed the presence of 37 phenolic compounds. Moreover, five compounds were isolated from the leaf extract, and identified on the basis of spectroscopic analysis. The isolated compounds were syringic acid (1), p-coumaric acid (2), 3',5' O-dicaffeoylquinic acid (3), luteolin-8-C-ß-D glucopyranoside (orientin) (4) and 8-methoxy kaempferol-3-O-[α-L-rhamnopyranosyl (1→2) ß-D-glucopyranoside] (5). The gentamicin-induced nephrotoxicity model was used to evaluate the protective effect of F. spragueana on renal toxicity biomarkers throughout the development of acute kidney injury. Administration of extract led to improvement in kidney function through inhibition of kidney injury molecule-1, creatinine, blood urea nitrogen and total bilirubin, as well as decreasing the inflammatory markers interlukin1-beta and myeloperoxidase. Furthermore, it reduced the oxidative stress by increasing reduced glutathione and total antioxidant capacity levels while decreasing malondialdehyde and nitric oxide content, and improved renal histopathological injuries.

Synthesis, molecular modeling studies, and anticonvulsant evaluation of novel 1-((2-hydroxyethyl)(aryl)amino)-N-substituted cycloalkanecarboxamides and their acetate esters.

A series of 1-((2-hydroxyethyl)(aryl)amino)-N-substituted cycloalkanecarboxamides IXa-l and their acetate esters Xa-l were designed and synthesized as new anticovulsant agents. The evaluation of the anticonvulsant effect was performed in vivo by subcutaneous pentylenetetrazole (scPTZ) and maximal electroshock (MES) tests in mice. Further, neurotoxicity, hepatotoxicity, and acute toxicity were determined. All the new candidates displayed 100% anticonvulsant activity in the scPTZ screen in the dose range of 0.0057-0.283 mmol/kg. The most potent compounds in the scPTZ screen were Xh (ED50 = 0.0012 mmol/kg), Xd (ED50 = 0.002 mmol/kg), Xf (ED50 = 0.004 mmol/kg), IXj (ED50 = 0.0047 mmol/kg), Xl (ED50 = 0.0076 mmol/kg), and Xi (ED50 = 0.008 mmol/kg). They exhibited higher fold activity in the anticonvulsant potential than the gold standards, phenobarbital and ethosuximide. Compound Xf was active in both scPTZ and MES screens. It showed ED50 of 0.016 mmol/kg in MES screen. In the neurotoxicity screens, none of the test compounds displayed any minimal motor impairment at the maximum administered dose. The 3D pharmacophore model using Biova 1 Discovery Studio 2016 programs exhibited high fit value. The anticonvulsant evaluation results were compatible with the molecular modeling study.

Assessment of anti-inflammatory, antinociceptive, immunomodulatory, and antioxidant activities of Cajanus cajan L. seeds cultivated in Egypt and its phytochemical composition.

CONTEXT: Cajanus cajan L. (Fabaceae), a food crop, is widely used in traditional medicine. OBJECTIVES: The phytochemical composition of C. cajan seeds and evaluation of the anti-inflammatory, immunomodulatory, antinociceptive, and antioxidant activities were studied. MATERIALS AND METHODS: Unsaponifiable matter and fatty acids were analyzed by GC and GC/MS. The n-butanol fraction was chromatographed on polyamide column. The anti-inflammatory activity of hexane extract (200 and 400 mg/kg, p.o.) was evaluated using the carrageenan-induced rat paw edema at 1, 2, and 3 h. The serum tumor necrosis factor-α, interleukin-6, and immunoglobulin G levels were detected by ELISA. The hexane extract antinociceptive activity was determined by adopting the writhing test in mice. DPPH radical scavenging, total reduction capability, and inhibition of lipid peroxidation of butanol fraction were evaluated. RESULTS AND CONCLUSION: Twenty-one unsaponifiable compounds (mainly phytol, 2,6-di-(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadiene-1-one, ß-sitosterol, stigmasterol, and campesterol), as well as 12 fatty acids (primarily 9,12-octadecadienoic and palmitic acids) were identified in hexane extract of C. cajan seeds. n-BuOH fraction contains quercetin-3-O-ß-d-glucopyranoside, orientin, vitexin, quercetin, luteolin, apigenin, and isorhamnetin. For the first time, quercetin-3-O-ß-d-glucopyranoside is isolated from C. cajan plant. The hexane extract (200 and 400 mg/kg) inhibited carrageenan-induced inflammation by 85 and 95%, respectively, 3 h post-carrageenan challenge. This was accompanied by an 11 and 20%, 8 and 13%, respectively, decrease of TNF-α and IL-6, as well as significant decrease in IgG serum levels. Moreover, hexane extract (200 and 400 mg/kg) decreased the number of writhings by 61 and 83%, respectively. The butanol fraction showed DPPH radical scavenging (inhibitory concentration (IC50) value: 9.07 µg/ml).

Anticonvulsant profiles of certain new 6-aryl-9-substituted-6,9-diazaspiro-[4.5]decane-8,10-diones and 1-aryl-4-substituted-1,4-diazaspiro[5.5]undecane-3,5-diones.

Synthesis and anticonvulsant potential of certain new 6-aryl-9-substituted-6,9-diazaspiro[4.5]decane-8,10-diones (6a-l) and 1-aryl-4-substituted-1,4-diazaspiro[5.5] undecane-3,5-diones (6m-x) are reported. The intermediates 1-[(aryl)(cyanomethyl)amino] cycloalkanecarboxamides (3a-f) were prepared via adopting Strecker synthesis on the proper cycloalkanone followed by partial hydrolysis of the obtained nitrile functionality and subsequent N-cyanomethylation. Compounds 3a-f were subjected to complete nitrile hydrolysis to give the respective carboxylic acid derivatives 4a-f which were cyclized under mild conditions to give the spiro compounds 5a-f. Ultimately, compounds 5a-f were alkylated or aralkylated to give the target compounds 6a-i and 6m-u. On the other hand, compounds 6j-l and 6v-x were synthesized from the intermediates 5a-f through alkylation, dehydration and finally tetrazole ring formation. Anticonvulsant screening of the target compounds 6a-x revealed that compound 6g showed an ED50 of 0.0043 mmol/kg in the scPTZ screen, being about 14 and 214 fold more potent than the reference drugs, Phenobarbital (ED50 = 0.06 mmol/kg) and Ethosuximide (ED50 = 0.92 mmol/kg), respectively. Compound 6e exhibited an ED50 of 0.019 mmol/kg, being about 1.8 fold more potent than that of the reference drug, Diphenylhydantoin (ED50 = 0.034 mmol/kg) in the MES screen. Interestingly, all the test compounds 6a-x did not show any minimal motor impairment at the maximum administered dose in the neurotoxicity screen.

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.

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.

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.

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.

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.

Constitutive expression and inducibility of CYP1A1 in the H9c2 rat cardiomyoblast cells.

Cardiomyocytes are a valuable tool for studying the drug metabolizing enzymes in the heart. However, isolated cardiomyocytes are rather fragile and difficult to isolate. Therefore, there is an urgent need for an in vitro cell line model. The H9c2 cells are commonly used as an in vitro model for studying the cellular mechanisms and signaling pathways involved in drug-induced cardiotoxicity. These cells maintain many molecular markers of cardiomyocytes and show morphological characteristics of immature embryonic cardiomyocytes. Therefore, in the present study we examined the expression and inducibility of CYP1A1 in the H9c2 rat cardiomyoblast cells. Our results showed that treatment of H9c2 cells with the CYP1A1 inducer, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly induced CYP1A1 at mRNA, protein, and activity levels in a concentration-dependent manner. The RNA synthesis inhibitor, actinomycin D, completely blocked the CYP1A1 mRNA induction by TCDD, indicating the requirement of de novo RNA synthesis through transcriptional activation. In conclusion, we demonstrated for the first time the constitutive expression and inducibility of CYP1A1 in H9c2 cells. Therefore, this cell line offers a unique in vitro model to study the role of CYP1A1 in the pathogenesis of various cardiovascular diseases.