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.

Blood pressure variability increases connexin expression in the vascular smooth muscle of rats

Aims Following sinoaortic denervation (SAD), isolated rat aortas present oscillatory contractions and demonstrate a heightened contraction for alpha-adrenergic agonists. Our aim was to verify the effects of SAD on connexin43 (Cx43) expression and phenylephrine-induced contraction in isolated aortas. Methods and results Three days after surgery (SAD or sham operation), isolated aortic rings were exposed to phenylephrine and acetylcholine (0.1-10 mu M) in the presence or absence of the gap junction blocker 18 beta-glycyrrhetinic acid (18 beta-GA, 100 mu M). Vascular reactivity to potassium chloride (KCl, 4.7-120 mM) was also examined. The incidence of rats presenting oscillatory contractions was measured. Effects of SAD on the vascular smooth muscle expression of the Cx43 mRNA by RT-PCR and western blotting for Cx43 protein were examined. Phenylephrine-induced contraction was higher in SAD rat aortas compared with the control. In the presence of 18 beta-GA, the response to phenylephrine was similar in both groups. Oscillatory contractions were observed in 10/10 SAD rat aortas vs. 2/10 controls. Relaxing response to acetylcholine was similar in both groups, but in the presence of 18 beta-GA, the response to acetylcholine decreased significantly in the sham-operated group (82.7 +/- 7.6% reduction of relaxation), whereas a half-maximal relaxation (reduction of 46.2 +/- 5.3%) took place in SAD rat aortas. KCl-induced contraction was similar in both groups. Following SAD, RT-PCR revealed significantly increased levels of Cx43 mRNA (9.85 fold, P < 0.01). Western blot analysis revealed greater levels of Cx43 protein (P < 0.05). Conclusion Blood pressure variability evoked by SAD leads to increased expression of Cx43, which could contribute to enhanced phenylephrine-induced contraction and oscillatory activity in isolated aortas.

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.

Determination of beta-artemether and its main metabolite dihydroartemisinin in plasma employing liquid-phase microextraction prior to liquid chromatographic-tandem mass spectrometric analysis

A method for the determination of artemether (ART) and its main metabolite dihydroartemisinin (DHA) in plasma employing liquid-phase microextraction (LPME) for sample preparation prior to liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed. The analytes were extracted from 1 nil, of plasma utilizing a two-phase LPME procedure with artemisinin as internal standard. Using the optimized LPME conditions, mean absolute recovery rates of 25 and 32% for DHA and ART, respectively, were achieved using toluene-n-octanol (1:1, viv) as organic phase with an extraction time of 30 min. After extraction, the analytes were resolved within 5 min using a mobile phase consisting of methanol-ammonium acetate (10 mmol L(-1) pH 5.0, 80:20. v/v) on a laboratory-made column based on poly(methyltetradecylsiloxane) attached to a zirconized-silica support. MS-MS detection was employed using an electrospray interface in the positive ion mode. The method developed was linear over the range of 5-1000 ng mL(-1) for both analytes. Precision and accuracy were within acceptable levels of confidence (<15%). The assay was applied to the determination of these analytes in plasma from rats treated with ART. The two-phase LPME procedure is affordable and the solvent consumption was very low compared to the traditional methods of sample preparation. (C) 2010 Elsevier B.V. All rights reserved.

Electrospray MS-based characterization of beta-carbolines - mutagenic constituents of thermally processed meat

The beta-carbolines 1-methyl-9H-pyrido [3,4-b]indole and 9H-pyrido[3,4b]indole have been implicated as having causative roles in a number of human diseases, such as Parkinson`s disease and cancer. As they can be formed during the heating of protein-rich food, a number of analytical methodologies have been proposed for their detection and quantification in foodstuff For this purpose, LC-MS and LC-MS/MS have emerged as the most specific analytical methods, and the quantification is based on the occurrence of unusual ions, such as [M+H-(H(center dot))](+) and [M + H-2H](+). In this study, we have investigated the formation of these ions by accurate-mass electrospray MS/MS and demonstrated that these ions are formed from gas-phase ion-molecule reactions between water vapor present in the collision cell and the protonated molecule of 1-methyl-9H-pyrido [3,4-b]indole and 9H-pyrido[3,4b]indole. Although this reaction has been previously described for heterocyclic amine ions, it has been overlooked in the most of recent LC-MS and LC-MS/MS studies, and no complete data of the fragmentation are reported. Our results demonstrate that additional attention should be given with respect to eliminating water vapor residues in the mass spectrometer when analysis of beta-carbolines is performed, as this residue may affect the reliability in the results of quantification.

Endogenous cannabinoids induce fever through the activation of CB(1) receptors

Background and purpose: The effects of centrally administered cannabinoids on body core temperature (Tc) and the contribution of endogenous cannabinoids to thermoregulation and fever induced by lipopolysaccharide (LPS) (Sigma Chem. Co., St. Louis, MO, USA) were investigated. Experimental approach: Drug-induced changes in Tc of male Wistar rats were recorded over 6 h using a thermistor probe (Yellow Springs Instruments 402, Dayton, OH, USA) inserted into the rectum. Key results: Injection of anandamide [(arachidonoylethanolamide (AEA); Tocris, Ellisville, MO, USA], 0.01-1 mu g i.c.v. or 0.1-100 ng intra-hypothalamic (i.h.), induced graded increases in Tc (peaks 1.5 and 1.6 degrees C at 4 h after 1 mu g i.c.v. or 10 ng i.h.). The effect of AEA (1 mu g, i.c.v.) was preceded by decreases in tail skin temperature and heat loss index (values at 1.5 h: vehicle 0.62, AEA 0.48). Bell-shaped curves were obtained for the increase in Tc induced by the fatty acid amide hydrolase inhibitor [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (Cayman Chemical Co., Ann Arbor, MI, USA) (0.001-1 ng i.c.v.; peak 1.9 degrees C at 5 h after 0.1 ng) and arachidonyl-2-chloroethylamide (ACEA; Tocris) (selective CB(1) agonist; 0.001-1 mu g i.c.v.; peak 1.4 degrees C 5 h after 0.01 mu g), but (R,S)-(+)-(2-Iodo-5-nitrobenzoyl)-[1-(1-methyl-piperidin-2-ylmethyl)-1H-indole-3-yl] methanone (Tocris) (selective CB(2) agonist) had no effect on Tc. AEA-induced fever was unaffected by i.c.v. pretreatment with 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indole-3-yl](4-methoxyphenyl) methanone (Tocris) (selective CB(2) antagonist), but reduced by i.c.v. pretreatment with N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; Tocris) (selective CB(1) antagonist). AM251 also reduced the fever induced by ACEA or LPS. Conclusions and implications: The endogenous cannabinoid AEA induces an integrated febrile response through activation of CB(1) receptors. Endocannabinoids participate in the development of the febrile response to LPS constituting a target for antipyretic therapy.

Preparation and characterization of D, L-PLA loaded 17-beta-Estradiol valerate by emulsion/evaporation methods

PLA microparticles containing 17-beta-estradiol valerate were prepared by an emulsion/evaporation method in order to sustain drug release. This system was characterized concerning particle size, particle morphology and the influence of formulation and processing parameters on drug encapsulation and in vitro drug release. The biodegradation of the microparticles was observed by tissue histological analysis. Scanning electron microscopy and particle size analysis showed that the microparticles were spherical, presenting non-aggregated homogeneous surface and had diameters in the range of 718-880 nm (inert microparticles) and 3-4 mu m (drug loaded microparticles). The encapsulation efficiency was similar to 80%. Hormone released from microparticles was sustained. An in vivo degradation experiment confirmed that microparticles are biodegradable. The preparation method was shown to be suitable, since the morphological characteristics and efficiency yield were satisfactory. Thus, the method of developed microparticles seems to be a promising system for sustained release of 17-beta-estradiol.

Production and characteristics comparison of crude beta-glucosidases produced by microorganisms Thermoascus aurantiacus e Aureobasidium pullulans in agricultural wastes

The purified beta-glucosidase of Aureobasidium pullulans ER-16 is one of more thermostable enzyme reported to date. Considering the unfeasibility of using purified enzyme for industrial application, it was interesting to analyze this property for the crude enzyme. Thermophilic fungus Thermoascus aurantiacus CBMAI-756 and mesophilic A. pullulans ER-16 were cultivated in different hemicellulosic materials on solid-state cultivation for beta-glucosidase production. Wheat bran was most appropriate for beta-glucosidase production by both microorganisms. T. aurantiacus exhibited maximum enzyme production (7.0 U/ml or 70 U/g) at 48-72 h and A. pullulans a maximum (1.3 U/ml or 13 U/g) at 120 h. Maximum activities were at 75 degrees C with optimum pH at 4.5 and 4.0, for T aurantiacus and A. pullulans, respectively. A. pullulans`s beta-glucosidase was more pH stable (4.5-10.0 against 4.5-8.0) and more thermostable (90% after 1 h at 75 degrees C against 85% after 1 h at 70 degrees C) than the enzyme from the thermophilic T. aurantiacus. The t((1/2)) at 80 degrees C were 50 and 12.5 min for A. pullulans and T. aurantiascus, respectively. These data confirm the high thermostability of crude beta-glucosidase from A. pullulans. Both beta-glucosidases were strongly inhibited by glucose, but ethanol significantly increased the activity of the enzyme from T. aurantiacus. (C) 2008 Elsevier Inc. All rights reserved.

Quercetin inhibits UV irradiation-induced inflammatory cytokine production in primary human keratinocytes by suppressing NF-kappa B pathway

Background: Topical flavonoids, such as quercetin, have been shown to reduce ultraviolet (UV) irradiation-mediated skin damage. However, the mechanisms and signaling pathways involved in this protective effect are not clear. UV irradiation leads to activation of two major signaling pathways, namely nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) pathways. Activation of NF-kappa B pathway by UV irradiation stimulates inflammatory cytokine expression, whereas activation of AP-1 pathway by UV irradiation promotes matrix metalloproteinase (MMP) production. Both pathways contribute to UV irradiation-induced skin damage, such as photoaging and skin tumor formation. Objective: To elucidate the underlying mechanism, we examined the effect of quercetin on UV irradiation induced activation of NF-kappa B and AP-1 pathways. Methods: Primary human keratinocytes, the major skin cell type subjected to physiological solar UV irradiation, were used to study the effects of quercetin on UV irradiation-induced signal transduction pathways. Results: Quercetin decreased UV irradiation-induced NF-kappa B DNA-binding by 80%. Consequently, quercetin suppressed UV irradiation-induced expression of inflammatory cytokines IL-1 beta (similar to 60%), IL-6 (similar to 80%), IL-8 (similar to 76%) and TNF-alpha (similar to 69%). In contrast, quercetin had no effect on UV irradiation activation of three MAP kinases, ERK, JNK, or p38. Accordingly, induction of AP-1 target genes such as MMP-1 and MMP-3 by UV irradiation was not suppressed by quercetin. Conclusion: Our data indicate that the ability of quercetin to block UV irradiation-induced skin inflammation is mediated, at least in part, by its inhibitory effect on NF-kappa B activation and inflammatory cytokine production. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

ATP-induced apoptosis involves a Ca(2+)-independent phospholipase A(2) and 5-lipoxygenase in macrophages

Macrophages express P2X(7) and other nucleotide (P2) receptors, and display the phenomena of extracellular ATP (ATP(e))-induced P2X(7)-dependent membrane permeabilization and cell death by apoptosis and necrosis. P2X7 receptors also cooperate with toll-like receptors (TLRs) to induce inflammasome activation and IL-1 beta secretion. We investigated signaling pathways involved in the induction of cell death by ATP, in intraperitoneal murine macrophages. Apoptosis (hypodiploid nuclei) and necrosis (LDH release) were detected 6 h after an induction period of 20 min in the presence of ATP Apoptosis was blocked by caspase 3 and caspase 9 inhibitors and by cyclosporin A. The MAPK inhibitors PD-98059, SB-203580 and SB-202190 provoked no significant effect oil apoptosis, but SB-203580 blocked LDH release. Neither apoptosis nor necrosis was inhibited when both intra- and extracellular Ca(2+) were chelated during the induction period. Mepacrine, a generic PLA(2) inhibitor and BEL, an inhibitor of Ca(2+)-independent PLA(2) (iPLA(2)) blocked apoptosis, while pBPB and AACOOPF(3). inhibitors of secretory and Ca(2+)-dependent PLA(2) respectively, had no significant effect. Cycloxygenase inhibitors had no effect on apoptosis, while the inhibitors of lipoxygenase (LOX) and leukotriene biosynthesis nordihydroguaiaretic acid (NDGA), zileuton, AA-861, and MK-886 significantly decreased apoptosis. Neither NDGA nor MK-886 blocked apoptosis of 5-LOX(-/-) macrophages. CP-105696 and MK-571, antagonists of leukotriene receptors, had no significant effect on apoptosis. None of the inhibitors of PLA(2) and LOX/leukotriene pathway had a significant inhibitory effect on LDH release. Our results indicate that a Ca(2+) -independent step involving an iPLA(2) and 5-LOX are involved in the triggering of apoptosis but not necrosis by P2X7 in macrophages. (C) 2008 Elsevier Inc. All rights reserved.

Suppressive action of melatonin on the TH-2 immune response in rats infected with Trypanosoma cruzi

Control of the acute phase of Trypanosoma cruzi infection is critically dependent on cytokine-mediated macrophage activation to intracellular killing, natural killer (NK) cells, CD4(+) T cells, CD8(+) T cells and B cells. Cell-mediated immunity in T. cruzi infection is also modulated by cytokines, but in addition to parasite-specific responses, autoimmunity can be also triggered. Importantly, cytokines may also play a role in the cell-mediated immunity of infected subjects. Here we studied the role of cytokines in the regulation of innate and adaptive immunity during the acute phase of T. cruzi infection in Wistar rats. Melatonin is an effective regulator of the immune system. Macrophages and T lymphocytes, which have melatonin receptors, are target cells for the immunomodulatory function of melatonin. In this paper melatonin was orally given via two protocols: prior to and concomitant with infection. Both treatments were highly effective against T. cruzi with enhanced action for the concomitant treatment. The data suggest an up-regulation of the TH-1 immune response as all analyzed parameters, interleukin (IL)-4, IL-10, transforming growth factor-beta 1 and splenocyte proliferation, displayed reduced levels as compared with the untreated counterparts. However, the direct effects of melatonin on immune cells have not been fully investigated during T. cruzi infection. We conclude that in light of the current results, melatonin exerted important therapeutic benefits through its immune regulatory effects.

Determinants of beta-lactam resistance in meningitis-causing Enterobacteriaceae in Brazil

This study analyzed resistance determinants in extended-spectrum beta-lactamase (ESBL)-producing enterobacteria and the epidemiology of 11 Escherichia coli isolates obtained from meningitis patients in a region of Brazil from 2000 to 2005. ESBL-encoding genes and their genetic environment were investigated by PCR and sequencing. The gene bla(CTX-M-2) was identified in 3 different enterobacteria (E. coli. Serratia marcescens, and Proteus mirabilis) downstream of the insertion sequence ISCR1 (localized in class 1 integrons), hut not as part of the resistance cassettes region. Multi locus sequence typing (MLST) was used to investigate genetic relationships between the 11 E. coil isolates in this study and strains associated with meningitis in the E. coil MLST database. MLST analysis indicated high genetic diversity among isolates, and no significant genetic relationship was identified with meningitis-causing E. coil in the database. The results in this report reinforce the need to be attentive to meningitis suspected to be due to ESBL-producing enterobacterial isolates, especially where ESBL epidemiology is well known.

Predominance of CTX-M-type extended-spectrum beta-lactamase genes among enterobacterial isolates from outpatients in Brazil

Two hundred fifty-seven nalidixic acid-resistant enterobacterial isolates were collected in a Brazilian community from January 2000 to May 2005 to determine the prevalence of plasmid-encoded extended-spectrum beta-lactamases. The bla(CTX-M) genetic environment was determined by polymerase chain reaction and sequencing. Eleven isolates (4.2%) harbored a bla(CTX-M-2) gene, 3 isolates bla(CTX-M-9), 2 isolates bla(CTX-M-8), and 6 isolates bla(SHV-5). Two novel bla(CTX-M-2) variants, namely, bla(CTX-M-74) and bla(CTX-M-75), were identified. (C) 2009 Elsevier Inc. All rights reserved.

Nosocomial outbreak by imipenem-resistant metallo-beta-lactamase-producing Pseudomonas aeruginosa in an adult intensive care unit in a Brazilian teaching hospital

Objective: To describe an outbreak of imipenem-resistant metallo-beta-lactamase-producing Pseudomonas aeruginosa, enzyme type bla, by horizontal transmission in patients admitted to a mixed adult ICU. Methods: A case-control study was carried out, including 47 patients (cases) and 122 patients (control) admitted to the mixed ICU of a university hospital in Minas Gerais. Brazil from November 2003 to July 2005. The infection site, risk factors, mortality, antibiotic susceptibility, metallo-beta-lactamase (MBL) production, enzyme type, and clonal diversity were analyzed, Results: A temporal/spatial relationship was detected in most patients (94%), overall mortality was 55.3%, and pneumonia was the predominant infection (85%). The majority of isolates (95%) were resistant to imipenem and other antibiotics, except for polymyxin, and showed MBL production (76.7%). Only bla SPM-1 (33%) was identified in the 15 specimens analyzed. In addition, 4 clones were identified, with a predominance of clone A (61.5%) and B (23.1%). On multivariate analysis, advanced age, mechanical ventilation, tracheostomy, and previous imipenem use were significant risk factors for imipenem-resistant P. aeruginosa infection. Conclusions: Clonal dissemination of MBL-producing P. aeruginosa strains with a spatial/temporal relationship disclosed problems in the practice of hospital infection control, low adherence to hand hygiene, and empirical antibiotic use. (C) 2008 Elsevier Espana, S.L. All rights reserved.

Synthesis of (2S)-isopropyl-5-alkynylpyrimidin-2-ones: precursors of beta-aminoacids

The efficient palladium-catalyzed Suzuki-Miyaura cross-coupling reaction of (2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-one with, arylethynyl-, heteroarylethynyl-, and alkylethynyltrifluoroborate salts is reported. The standard protocol was evaluated and optimized in order to gain access to suitable precursors of enantiopure 2-substituted beta-amino acids. The scope and limitations of this methodology are discussed. (C) 2010 Elsevier Ltd. All rights reserved.