Cruzain inhibition by hydroxymethylnitrofurazone and nitrofurazone: investigation of a new target in Trypanosoma cruzi

Nitrofurazone (NF) and its derivative, hydroxymethylnitrofurazone (NFOH), have presented antichagasic activity. NFOH has higher activity and lower mutagenicity. The aim of this work was to assess whether NF and its derivative NFOH would also be inhibitors of cruzain, besides their trypanothione reductase inhibitory activity. In vitro cruzain inhibition tests were performed for both compounds, and the 50% inhibitory concentration (IC(50)) for NF and NFOH presented values of 22.83 +/- 1.2 mu M and 10.55 +/- 0.81 mu M, respectively. AM1 semi-empirical molecular modeling studies were performed to understand the activity of the compounds, corroborating the observed cruzain inhibitory activity.

Chemopreventive effects of beta-ionone and geraniol during rat hepatocarcinogenesis promotion: distinct actions on cell proliferation, apoptosis, HMGCoA reductase, and RhoA

Chemopreventive activities of the dietary isoprenoids beta-ionone (beta I) and geraniol (GOH) were evaluated during the promotion phase of hepatocarcinogenesis. Over 5 consecutive weeks, rats received daily 16 mg/100 g body weight (b.w.) of beta I (beta I group), 25 mg/100 g b.w. of GOH (GOH group), or only corn oil (CO group, controls). Compared to the CO group, the following was observed: only the beta I group showed a decrease in the mean number of visible hepatocyte nodules (P<.05); beta I and GOH groups had reduced mean number of persistent preneoplastic lesions (pPNLs) (P<.05), but no differences regarding number of remodeling PNL (rPNLs) were observed; only the beta I group exhibited smaller rPNL size and percentage of liver sections occupied by pPNLs (P<.05), whereas the GOH group displayed a smaller percentage of liver sections occupied by rPNLs (P<.05); a trend was observed in the beta I group, which showed reduced cell proliferation of pPNLs (P<.10), and the GOH group had increased apoptosis in pPNLs and rPNLs (P<.05); only the beta I group displayed reduced total plasma cholesterol concentrations (P<.05) and increased hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase mRNA levels (P<.05): only the GOH group had lower hepatic membrane RhoA protein levels (P<.05); both the beta I- and GOH-treated groups had higher hepatic concentrations of beta I and GOH, respectively (P<.05). Given these data, beta I and GOH show promising chemopreventive effects during promotion of hepatocarcinogenesis by acting through distinct mechanism of actions: beta I may inhibit cell proliferation and modulate HMGCoA reductase, and GOH can induce apoptosis and inhibit RhoA activation. (C) 2011 Elsevier Inc. All rights reserved.

On the Cytoadhesion of Plasmodium vivax-Infected Erythrocytes

Background. Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. Methods. Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. Results. Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. Conclusions. These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

Chemoprevention of rat hepatocarcinogenesis with histone deacetylase inhibitors: Efficacy of tributyrin, a butyric acid prodrug

Hepatocellular carcinoma (HCC) ranks in prevalence and mortality among top 10 cancers worldwide. Butyric acid (BA), a member of histone deacetylase inhibitors (HDACi) has been proposed as an anticareinogenic agent. However, its short half-life is a therapeutical limitation. This problem could be circumvented with tributyrin (TB), a proposed BA prodrug. To investigate TB effectiveness for chemoprevention, rats were treated with the compound during initial phases of ""resistant hepatocyte"" model of hepatocarcinogenesis, and cellular and molecular parameters were evaluated. TB inhibited (p < 0.05) development of hepatic preneoplastic lesions (PNL) including persistent ones considered HCC progression sites. TB increased (p < 0.05) PNL remodeling, a process whereby they tend to disappear. TB did not inhibit cell proliferation in PNL, but induced (p < 0.05) apoptosis in remodeling ones. Compared to controls, rats treated with TB presented increased (P < 0.05) hepatic levels of BA indicating its effectiveness as a prodrug. Molecular mechanisms of TB-induced hepatocarcinogenesis chemoprevention were investigated. TB increased (p < 0.05) hepatic nuclear histone H3K9 hyperacetylation specifically in PNL and p21 protein expression, which could be associated with inhibitory HDAC effects. Moreover, it reduced (p < 0.05) the frequency of persistent PNL with aberrant cytoplasmic p53 accumulation, an alteration associated with increased malignancy. Original data observed in our study support the effectiveness of TB as a prodrug of BA and as an HDACi in hepatocarcinogenesis chemoprevention. Besides histone acetylation and p21 restored expression, molecular mechanisms involved with TB anticarcinogenic actions could also be related to modulation of p53 pathways. (C) 2008 Wiley-Liss, Inc.

Molecular modeling studies and in vitro bioactivity evaluation of a set of novel 5-nitro-heterocyclic derivatives as anti-T. cruzi agents

In this study, in vitro anti-T. cruzi activity assays of nifuroxazide (NX) analogues, such as 5-nitro-2-furfuryliden and 5-nitro-2-theniliden derivatives, were performed. A molecular modeling approach was also carried out to relate the lipophilicity potential ( LP) property and biological activity data. The majority of the NX derivatives showed increased anti-T. cruzi activity in comparison to the reference drug, benznidazole (BZN). Additionally, the 5-nitro-2-furfuryliden derivatives presented better pharmacological profile than the 5-nitro-2-theniliden analogues. The LP maps and corresponding ClogP values indicate that there is an optimum lipophilicity value, which must be observed in the design of new potential anti-T. cruzi agents. (c) 2009 Elsevier Ltd. All rights reserved.

In vitro characterization of rosiglitazone metabolites and determination of the kinetic parameters employing rat liver microsomal fraction

Rosiglitazone (RSG), a thiazolidinedione antidiabetic drug, is metabolized by CYP450 enzymes into two main metabolites: N-desmethyl rosiglitazone (N-Dm-R) and rho-hydroxy rosiglitazone (rho-OH-R). In humans, CYP2C8 appears to have a major role in RSG metabolism. On the other hand, the in vitro metabolism of RSG in animals has not been described in literature yet. Based on these concerns, the kinetic metabolism study of RSG using rat liver microsomal fraction is described for the first time. Maximum velocity (V (max)) values of 87.29 and 51.09 nmol/min/mg protein were observed for N-Dm-R and rho-OH-R, respectively. Michaelis-Menten constant (K (m)) values were of 58.12 and 78.52 mu M for N-Dm-R and rho-OH-R, respectively. Therefore, these results demonstrated that this in vitro metabolism model presents the capacity of forming higher levels of N-Dm-R than of rho-OH-R, which also happens in humans. Three other metabolites were identified employing mass spectrometry detection under positive electrospray ionization: ortho-hydroxy-rosiglitazone (omicron-OH-R) and two isomers of N-desmethyl hydroxy-rosiglitazone. These metabolites have also been observed in humans. The results observed in this study indicate that rats could be a satisfactory model for RSG metabolism.

Alprazolam potentiates the antiaversive effect induced by the activation of 5-HT(1A) and 5-HT(2A) receptors in the rat dorsal periaqueductal gray

Rationale Serotonin in the dorsal periaqueductal gray (DPAG) through the activation of 5-HT(1A) and 5-HT(2A) receptors inhibits escape, a defensive behavior associated with panic attacks. Long-term treatment with antipanic drugs that nonselectively or selectively blocks the reuptake of serotonin (e.g., imipramine and fluoxetine, respectively) enhances the inhibitory effect on escape caused by intra-DPAG injection of 5-HT(1A) and 5-HT(2A) receptor agonists. It has been proposed that these compounds exert their effect on panic by facilitating 5-HT-mediated neurotransmission in the DPAG. Objectives The objective of this study was to investigate whether facilitation of 5-HT neurotransmission in the DPAG is also observed after treatment with alprazolam, a pharmacologically distinct antipanic drug that acts primarily as a high potency benzodiazepine receptor agonist. Materials and methods Male Wistar rats, subchronically (3-6 days) or chronically (14-17 days) treated with alprazolam (2 and 4 mg/kg, i.p.) were intra-DPAG injected with (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), (+/-)-1-(2,5-dimethoxy-4-iodophenyl) piperazine dihydrochloride (DOI), and midazolam, respectively, 5-HT(1A), 5-HT(2A/2C), and benzodiazepine receptor agonists. The intensity of electrical current that needed to be applied to the DPAG to evoke escape behavior was measured before and after the microinjection of these agonists. Results Intra-DPAG injection of the 5-HT agonists and midazolam increased the escape threshold in all groups of animals tested, indicating a panicolytic-like effect. The inhibitory effect of 8-OH-DPAT and DOI, but not midazolam, was significantly higher in animals receiving long-, but not short-term treatment with alprazolam. Conclusions Alprazolam as antidepressants compounds facilitates 5-HT(1A)- and 5-HT(2A)-receptor-mediated neurotransmission in the DPAG, implicating this effect in the mode of action of different classes of antipanic drugs.

Crystal structure of Trypanosoma cruzi dihydroorotate dehydrogenase from Y strain

Trypanosoma cruzi is the etiological agent of Chagas` disease, a pathogenesis that affects millions of people in Latin America. Here, we report the crystal structure of dihydroorotate dehydrogenase (DHODH) from T cruzi strain Y solved at 2.2 angstrom resolution. DHODH is a flavin mononucleotide containing enzyme, which catalyses the oxidation Of L-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Genetic studies have shown that DHODH is essential for T cruzi survival, validating the idea that this enzyme can be considered an attractive target for the development of antichagasic drugs. In our work, a detailed analysis of T cruzi DHODH crystal structure has allowed us to suggest potential sites to be further exploited for the design of highly specific inhibitors through the technology of structure-based drug design. (c) 2008 Elsevier Inc. All rights reserved.

Protective effects of Mangifera indica L extract (Vimang), and its major component mangiferin, on iron-induced oxidative damage to rat serum and liver

In vivo preventive effects of a Mangifera indica L extract (Vimang) or its major component mangiferin on iron overload injury have been studied in rats given respectively, 50, 100, 250 mg kg(-1) body weight of Vimang, or 40 mg kg(-1) body weight of mangiferin, for 7 days prior to, and for 7 days following the administration of toxic amounts of iron-dextran. Both Vimang or mangiferin treatment prevented iron overload in serum as well as liver oxidative stress, decreased serum and liver lipid peroxidation, serum GPx activity, and increased serum and liver GSH, serum SOD and the animals overall antioxidant condition. Serum iron concentration was decreased although at higher doses, Vimang tended to increase it; percent tranferrin saturation, liver weight/body mass ratios, liver iron content was decreased. Treatment increased serum iron-binding capacity and decreased serum levels of aspartate-amine transferase (ASAT) and alanine-amine transferase (ALAT), as well as the number of abnormal Kupffer cells in iron-loaded livers. It is suggested that besides acting as antioxidants, Vimang extract or its mangiferin component decrease liver iron by increasing its excretion. Complementing earlier in vitro results from our group, it appears possible to support the hypothesis that Vimang and mangiferin present therapeutically useful effects in iron overload related diseases. (C) 2007 Elsevier Ltd. All rights reserved.

Balloon catheter injury abolishes phenylephrine-induced relaxation in the rat contralateral carotid

BACKGROUND AND PURPOSE The consequences of compensatory responses to balloon catheter injury in rat carotid artery, on phenylephrine-induced relaxation and contraction in the contralateral carotid artery were studied. EXPERIMENTAL APPROACH Relaxation and contraction concentration-response curves for phenylephrine were obtained for contralateral carotid arteries in the presence of indomethacin (COX inhibitor), SC560 (COX-1 inhibitor), SC236 (COX-2 inhibitor) or 4-hydroxytetramethyl-L-piperidine-1-oxyl (tempol; superoxide dismutase mimetic). Reactive oxygen species were measured in carotid artery endothelial cells fluorimetrically with dihydroethidium. KEY RESULTS Phenylephrine-induced relaxation was abolished in contralateral carotid arteries from operated rats (E(max) = 0.01 +/- 0.004 g) in relation to control (E(max) = 0.18 +/- 0.005 g). Phenylephrine-induced contractions were increased in contralateral arteries (E(max) = 0.54 +/- 0.009 g) in relation to control (E(max) = 0.38 +/- 0.014 g). SC236 restored phenylephrine-induced relaxation (E(max) = 0.17 +/- 0.004 g) and contraction (E(max) = 0.34 +/- 0.018 g) in contralateral arteries. Tempol restored phenylephrine-induced relaxation (E(max) = 0.19 +/- 0.012 g) and contraction (E(max) = 0.42 +/- 0.014 g) in contralateral arteries, while apocynin did not alter either relaxation (E(max) = 0.01 +/- 0.004 g) or contraction (E(max) = 0.54 +/- 0.009 g). Dihydroethidium fluorescence was increased in contralateral samples (18 882 +/- 435 U) in relation to control (10 455 +/- 303 U). SC236 reduced the fluorescence in contralateral samples (8250 +/- 365 U). CONCLUSIONS AND IMPLICATIONS Balloon catheter injury abolished phenylephrine-induced relaxation and increased phenylephrine-induced contraction in contralateral carotid arteries, through O(2)(-) derived from COX-2.

Acidosis induces relaxation mediated by nitric oxide and potassium channels in rat thoracic aorta

We investigated the mechanism by which extracellular acidification promotes relaxation in rat thoracic aorta. The relaxation response to HCl-induced extracellular acidification (7.4 to 6.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M) or KCl (45 mM). The vascular reactivity experiments were performed in endothelium-intact and denuded rings, in the presence or absence of indomethacin (10(-5) M), L-NAME (10(-4) M), apamin (10(-6) M), and glibenclamide (10(-5) M). The effect of extracellular acidosis (pH 7.0 and 6.5) on nitric oxide (NO) production was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 mu M). The extracellular acidosis failed to induce any changes in the vascular tone of aortic rings pre-contracted with KCl, however, it caused endothelium-dependent and independent relaxation in rings pre-contracted with Phe. This acidosis induced-relaxation was inhibited by L-NAME, apamin, and glibenclamide, but not by indomethacin. The acidosis (pH 7.0 and 6.5) also promoted a time-dependent increase in the NO production by the isolated endothelial cells. These results suggest that extracellular acidosis promotes vasodilation mediated by NO, K(ATP) and SK(Ca), and maybe other K(+) channels in isolated rat thoracic aorta. (C) 2011 Elsevier B.V. All rights reserved.

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta

Aim: To investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta. Methods: The relaxation response to NaOH-induced extracellular alkalinization (7.4-8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10(-5) M), NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10(-7) M), 2,5-dimethylbenzimidazole (DMB, 2 x 10(-5) M) and methyl-B-cyclodextrin (10(-2) M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 mu M), in the presence and absence of DMB (2 x 10(-5) M). Results: The extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca(2+)/calmodulin and Na(+)/Ca(2+). exchanger (NCX), and partially blunted by the caveolae disassembly. Conclusions: These results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca(2+) concentration and activating the Ca(2+)/calmodulin-dependent NOS. In turn, NO is released promoting relaxation. (C) 2010 Elsevier Inc. All rights reserved.

Simultaneous determination of rosiglitazone and its metabolites in rat liver microsomal fraction using hollow-fiber liquid-phase microextraction for sample preparation

A three-phase hollow-fiber liquid-phase microextraction method for the analysis of rosiglitazone and its metabolites N-desmethyl rosiglitazone and p-hydroxy rosiglitazone in microsomal preparations is described for the first time. The drug and metabolites HPLC determination was carried out using an X-Terra RP-18 column, at 22 degrees C. The mobile phase was composed of water, acetonitrile and acetic acid (85:15:0.5, v/v/v) and the detection was performed at 245 nm. The hollow-fiber liquid-phase microextraction procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1750 rpm, extraction for 30 min, hydrochloric acid 0.01 mol/L as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 8.0. The recovery rates, obtained by using 1 mL of microsomal preparation, were 47-70%. The method presented LOQs of 50 ng/mL and it was linear over the concentration range of 50-6000 ng/mL, with correlation coefficients (r) higher than 0.9960, for all analytes. The validated method was employed to study the in vitro biotransformation of rosiglitazone using rat liver microsomal fraction.

LC-MS-MS Identification and Determination of the Flavone-C-Glucoside Vicenin-2 in Rat Plasma Samples Following Intraperitoneal Administration of Lychnophora Extract

The flavone C-glucoside, vicenin-2, in semi-purified extracts of the leaves of Lychnophora ericoides was quantified in rat plasma samples using a method based on reversed-phase high performance liquid chromatography coupled to tandem mass spectrometry. Vicenin-2 was analyzed on a LiChrospher (R) RP18 column using an isocratic mobile phase consisting of a mixture of methanol: water (30:70, v/v) plus 2.0% glacial acetic acid at a flow rate of 0.8 mL min(-1). Genistein was used as internal standard. The mass spectrometer was operated in positive ionization mode and analytes were quantified by multiple reaction monitoring at m/z 595 > 457 for vicenin-2 and m/z 271 > 153 for internal standard. Prior to the analysis, each rat plasma sample was acidified with 200 mu L of 50 mmol L(-1) acetic acid solution and extracted by solid-phase extraction using a C18 cartridge. The absolute recoveries were reproducible and the coefficients of variation values were lower than 5.2%. The method was linear over the 12.5 - 1500 ng mL(-1) concentration range and the quantification limit was 12.5 ng mL(-1). Within-day and between-day assay precision and accuracy were studied at three concentration levels (40, 400 and 800 ng mL(-1)) and were lower than 15%. The developed and validated method seems to be suitable for analysis of vicenin-2 in plasma samples obtained from rats that receive a single i.p. dose of 200 mg kg(-1) vicenin-2 extract.

Chronic hyperhomocysteinemia impairs vascular function in ovariectomized rat carotid arteries

Homocysteine is an independent risk factor for coronary heart disease, as well as for cerebrovascular and peripheral vascular diseases. The purpose of this study was to investigate the effects of hyperhomocysteinemia (HHcy) on vascular reactivity within carotid artery segments isolated from ovariectomized female rats. Treatment with dl-Hcy thiolactone (1 g/kg body weight per day) reduced the phenylephrine-induced contraction of denuded rings. However, the treatment did not alter KCl-induced contractions, or relaxations induced by sodium nitroprusside or acetylcholine. We report elevated expressions of iNOS, eNOS, and nitrotyrosine in homocysteine-treated rat artery sections. Moreover, the inhibition of NOS by l-NAME, 1,400 W, or l-NNA restored phenylephrine-induced vasoconstriction in carotid artery segments from Hcy-treated rats. In conclusion, our findings show that severe HHCy can promote an acute decrease in the endothelium-independent contractile responses of carotid arteries to adrenergic agonists. This effect was restored by nitric oxide synthase inhibitors, which further supports the involvement of nitric oxide in HHcy-derived vascular dysfunction.