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.

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 mu M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca(2+) efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP(+) transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. (C) 2011 Elsevier Inc. All rights reserved.

The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 mu M) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Fluoxetine induces preventive and complex effects against colon cancer development in epithelial and stromal areas in rats

Fluoxetine (FIX) is a drug commonly used as antidepressant. However, its effects on tumorigenesis remain controversial. Aiming to evaluate the effects of FIX treatment on early malignant changes, we analyzed serotonin (5-HT) metabolism and recognition, aberrant crypt foci (ACF), proliferative process, microvessels, vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2) expression in colon tissue. Male Wistar rats received a daily FLX-gavage (30 mg kg(-1)) and, a single dose of 1.2 dimethylhydrazine (DMH; i.p., 125 mg kg(-1)). After 6 weeks of FIX-treatment, our results revealed that FIX and nor-fluoxetine (N-FIX) are present in colon tissue, which was related to significant increase in serotonin (5-HT) levels (P < 0.05) possibly through a blockade in SERT mRNA (serotonin reuptake transporter; P < 0.05) resulting in lower 5-hydroxyindoleacetic acid (5-HIAA) levels (P < 0.01) and, 5-HT2C receptor mRNA expressions. FIX-treatment decreased dysplastic ACF development (P < 0.01) and proliferative process (P < 0.001) in epithelia. We observed a significant decrease in the development of malignant microvessels (P < 0.05), VEGF (P < 0.001), and COX-2 expression (P < 0.01). These findings suggest that FIX may have oncostatic effects on carcinogenic colon tissue, probably due to its modulatory activity on 5-HT metabolism and/or its ability to reduce colonic malignant events. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Molecular dynamics, density functional, ADMET predictions, virtual screening, and molecular interaction field studies for identification and evaluation of novel potential CDK2 inhibitors in cancer therapy

In this work, we have used molecular dynamics, density functional theory, virtual screening, ADMET predictions, and molecular interaction field studies to design and propose eight novel potential inhibitors of CDK2. The eight molecules proposed showed interesting structural characteristics that are required for inhibiting the CDK2 activity and show potential as drug candidates for the treatment of cancer. The parameters related to the Rule of Five were calculated, and only one of the molecules violated more than one parameter. One of the proposals and one of the drug-like compounds selected by virtual screening indicated to be promising candidates for CDK2-based cancer therapy.

In vitro studies of cutaneous retention of magnetic nanoemulsion loaded with zinc phthalocyanine for synergic use in skin cancer treatment

In this study was developed a new nano drug delivery system (NDDS) based on association of biodegradable surfactants with biocompatible magnetic fluid of maguemita citrate derivative. This formulation consists in a magnetic emulsion with nanostructured colloidal particles. Preliminary in vitro experiments showed that the formulation presents a great potential for synergic application in the topical release of photosensitizer drug (PS) and excellent target tissue properties in the photodynamic therapy (PDT) combined with hyperthermia (HPT) protocols. The physical chemistry characterization and in vitro assays were carried out by Zn(II) Phtalocyanine (ZnPc) photosensitizer incorporated into NDDS in the absence and the presence of magnetic fluid, showed good results and high biocompatibility. In vitro experiments were accomplished by tape-stripping protocols for quanti. cation of drug association with different skin tissue layers. This technique is a classical method for analyses of drug release in stratum corneum and epidermis+ dermis skin layers. The NDDS formulations were applied directly in pig skin (tissue model) fixed in the cell`s Franz device with receptor medium container with a PBS/EtOH 20% solution (10mM, pH 7.4) at 37 degrees C. After 12 h of topical administration stratum corneum was removed from fifty tapes and the ZnPc retained was evaluated by solvent extraction in dimetil-sulphoxide under ultrasonic bath. These results indicated that magnetic nanoemulsion (MNE) increase the drug release on the deeper skin layers when compared with classical formulation in the absence of magnetic particles. This could be related with the increase of biocompatibility of NDDS due to the great affinity for the polar extracelullar matrix in the skin and also for the increase in the drug partition inside of corneocites wall. (C) 2008 Elsevier B.V. All rights reserved.

Photophysical studies and in vitro skin permeation/retention of Foscan (R)/nanoemulsion (NE) applicable to photodynamic therapy skin cancer treatment.

In this work we evaluated the photophysical and in vitro properties of Foscan (R), a second-generation photosensitizer drug (PS) widely used in systemic clinical protocols for cancer therapy based on Photodynamic Therapy (PDT). We employed biodegradable nanoemulsions (NE) as a colloidal vehicle of the oil/water (o/w) type focusing in topical administration of Foscan (R) and other photosensitizer drugs. This formulation was obtained and stabilized by the methodology described by Tabosa do Egito et al.,(30) based on the mixture of two phases: an aqueous solution and an organic medium consisting of nonionic surfactants and oil. The photodynamic potential of the drug incorporated into the NE was studied by steady-state and time-resolved spectroscopic techniques. We also analyzed the in vitro biological behavior carried out in mimetic biological environment protocols based on the animal model. After topical application in a skin animal model, we evaluated the Foscan (R)/NE diffusion flux into the skin layers (stratum corneum and epidermis + dermis) by classical procedures using Franz Diffusion cells. Our results showed that the photophysical properties of PS were maintained after its incorporation into the NE when compared with homogeneous organic medium. The in vitro assays enabled the determination of an adequate profile for the interaction of this system in the different skin layers, with an ideal time lag of 6 h after topical administration in the skin model. The Foscan (R) diffusion flux (J) was increased when this PS was incorporated into the NE, if compared with its flux in physiological medium. These parameters demonstrated that the NE can be potentially applied as a drug delivery system (DDS) for Foscan (R) in both in vitro and in vivo assays, as well as in future clinical applications involving topical skin cancer PDT.

Fluoride increases lead concentrations in whole blood and in calcified tissues from lead-exposed rats

Higher blood lead (BPb) levels have been reported in children living in communities that receive fluoride-treated water. Here, we examined whether fluoride co-administered with lead increases BPb and lead concentrations in calcified tissues in Wistar rats exposed to this metal from the beginning of gestation. We exposed female rats and their offspring to control water (Control Group), 100 mg/L of fluoride (F Group), 30 mg/L of lead (Pb Group), or 100 mg/L of fluoride and 30 mg/L of lead (F+ Pb Group) from 1 week prior to mating until offspring was 81 days old. Blood and calcified tissues (enamel, dentine, and bone) were harvested at day 81 for lead and fluoride analyses. Higher BPb concentrations were found in the F+ Pb Group compared with the Pb Group (76.7 +/- 11.0 mu g/dL vs. 22.6 +/- 8.5 mu g/dL, respectively: p <0.001). Two-to threefold higher lead concentrations were found in the calcified tissues in the F+ Pb Group compared with the Pb Group (all p <0.001). Fluoride concentrations were similar in the F and in the F+ Pb Groups. These findings show that fluoride consistently increases BPb and calcified tissues Pb concentrations in animals exposed to low levels of lead and suggest that a biological effect not yet recognized may underlie the epidemiological association between increased BPb lead levels in children living in water-fluoridated communities. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Methylmercury and inorganic mercury determination in blood by using liquid chromatography with inductively coupled plasma mass spectrometry and a fast sample preparation procedure

Despite the necessity to differentiate chemical species of mercury in clinical specimens, there area limited number of methods for this purpose. Then, this paper describes a simple method for the determination of methylmercury and inorganic mercury in blood by using liquid chromatography with inductively coupled mass spectrometry (LC-ICP-MS) and a fast sample preparation procedure. Prior to analysis, blood (250 mu L) is accurately weighed into 15-mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCI was added to the samples following sonication for 15 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of mercury species was accomplished in less than 5 min on a C18 reverse-phase column with a mobile phase containing 0.05% (v/v) mercaptoethanol, 0.4% (m/v) L-cysteine, 0.06 mol L(-1) ammonium acetate and 5% (v/v) methanol. The method detection limits were found to be 0.25 mu g L(-1) and 0.1 mu Lg L(-1) for inorganic mercury and methylmercury, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). The proposed method was also applied to the speciation of mercury in blood samples collected from fish-eating communities and from rats exposed to thimerosal. With the proposed method there is a considerable reduction of the time of sample preparation prior to speciation of Hg by LC-ICP-MS. Finally, after the application of the proposed method, we demonstrated an interesting in vivo ethylmercury conversion to inorganic mercury. (C) 2009 Elsevier B.V. All rights reserved.

Direct and Simultaneous Determination of Cd Cu, and Se in Blood Samples by Graphite Furnace Atomic Absorption Spectrometry

A graphite furnace atomic absorption spectrometric method is proposed for the direct and simultaneous determination of Cd, Cu, and Se in human blood. Samples were diluted 1:10 (v/v) in 0.5% (v/v) HNO(3) + 0.5% (v/v) Triton X-100 solution. For 12 mu L injected sample volume + 5 mu L, of 1000 mg L(-1) Pd(NO(3))(2) + 3 mu L of 1000 mg L(-1) Mg(NO(3))(2), the calculated characteristic masses (mo) were 0.9 pg Cd, 16 pg Cu, and 39 pg Se, which are close to those mo values for single-element conditions for THGA furnace (1.3 pg Cd, 17 pg Cu, and 45 pg Se). Calibration curves with linear correlations better than 0.999 were obtained. The limits of detection (LOD) were 0.03 mu g L(-1) Cd, 0.075 mu g L(-1) Cu and 0.3 mu g L(-1) Se, and the relative standard deviations (n= 12) were 2.5%, 0.3%, and 1.5%, respectively. The method was applied for Cd, Cu, and Se determination in 10 human blood samples and the results were in agreement at the 95% confidence level with those obtained by inductively coupled plasma mass spectrometry. Concentrations of analytes in the selected blood samples varied from 1.7 to 3.2 mu g L(-1) Cd, 700 to 921.7 mu g L(-1) Cu, and from 68.6 to 350 mu g L(-1) Se. The accuracy of the proposed method was also evaluated by an addition-recovery experiment and recoveries of Cd, Cu, and Se added to blood samples ranged from 99-109%, 91-103%,and 93-103%, respectively.

Elevated blood lead levels in a riverside population in the Brazilian Amazon

Lead (Pb) is recognized as one of the most toxic metals. Sources of Pb exposure have been widely documented in North America, and the removal of Pb additives from gasoline was reflected in a dramatic lowering of blood Pb concentration. In Latin America, the removal of Pb from gasoline resulted in decreased exposure, but Pb levels in many areas remain high due to occupational and environmental sources of exposure. While many of the Pb sources have been identified (mining, industries, battery recycling, lead-based paint, ceramics), new ones occasionally crop up. Here we report on blood Pb (B-Pb) levels in remote riverside communities of the Brazilian Amazon. Blood Pb (B-Pb) levels were determined in 448 persons from 12 villages of the Lower Tapajos River Basin, Par, Brazil. Socio-demographic and dietary information, as well as occupational, residential and medical history was collected using an interview-administered questionnaire. B-Pb, measured by ICP-MS, showed elevated concentrations. Mean B-Pb was 13.1 mu g/dL +/- 8.5, median B-Pb was 11.2 mu g/dL and ranged from 0.59 to 48.3 mu g/dL. Men had higher B-Pb compared to women (median: 15.3 mu g/dL vs 7.9 mu g/dL respectively). B-Pb increased with age for women, while it decreased for men. For both genders, B-Pb decreased with education. There were significant differences between villages. Exploratory analyses, using linear partition models, showed that for men B-Pb was lower among those who were involved in cattle-raising, and higher among those who hunted, farmed and fished. The distribution profile of B-Pb directed us towards artisanal transformation of manioc to flour (farinha), which requires heating in a large metal pan, with stirring primarily done by young men. In the village with the highest B-Pb, analysis of Pb concentrations (dry weight) of manioc (prior to transformation) and farinha (following transformation) from 6 houses showed a tenfold increase in Pb concentration (mean: 0.017 +/- 0.016 to 0.19 +/- 0.10 mu g/g). This was confirmed in one of these villages where we sampled manioc paste Oust before roasting) and the roasted farinha (0.05 mu g/g vs 0.20 mu g/g). While there may be other sources (ammunition, sinkers for fishing nets), the high concentrations in farinha, a dietary staple, assuredly makes an important contribution. Further action needs to reduce Pb sources in this region. (C) 2009 Elsevier Inc. All rights reserved.

Determination of total and inorganic mercury in whole blood by cold vapor inductively coupled plasma mass spectrometry (CV ICP-MS) with alkaline sample preparation

A simple method with a fast sample preparation procedure for total and inorganic mercury determinations in blood samples is proposed based on flow injection cold vapor inductively coupled plasma mass spectrometry (FI-CVICP-MS). Aliquots of whole blood (500 mL) are diluted 1 + 1 v/v with 10.0% v/v tetramethylammonium hydroxide (TMAH) solution, incubated for 3 h at room temperature and then further diluted 1 + 4 v/v with 2.0% v/v HCl. The inorganic Hg was released by online addition of L-cysteine and then reduced to elemental Hg by SnCl(2). On the other hand, total mercury was determined by on-line addition of KMnO(4) and then reduced to elemental Hg by NaBH(4). Samples were calibrated against matrix-matching. The method detection limit was found to be 0.80 mu g L(-1) and 0.08 mu g L(-1) for inorganic and total mercury, respectively. Sample throughput is 20 samples h(-1). The method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). For additional validation purposes, human whole blood samples were analyzed by the proposed method and by an established CV AAS method, with no statistical difference between the two techniques at 95% confidence level on applying the t-test.

Assessment of how pregnancy modifies plasma lead and plasma/whole blood lead ratio in ALAD 1-1 genotype women

Pregnant women are one of the most sensitive populations to the toxic effects associated with lead (Pb) exposure. These effects are primarily associated with plasma Pb (Pb-P), which reflects the most rapidly exchangeable fraction of Pb in the bloodstream, and elevated maternal Pb-P may be more relevant to foetal Pb exposure than whole blood Pb (Pb-B). We investigated how pregnancy affects Pb-B, Pb-P and %Pb-P/Pb-B ratios without the influence of the 6-aminolevulinic acid dehydratase (ALAD) G177C polymorphism, which is a major genetic factor influencing Pb-B, Pb-P and %Pb-P/Pb-B ratios. Genotypes for the ALAD G177C polymorphism were determined by PCR and restriction fragment length digestion in nine pregnant and 20 non-pregnant women, aged 18-33, environmentally exposed to Pb. Here, we included only women with ALAD 1-1 genotype. Pb-P and Pb-B were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. We found no differences in Pb-B (P > 0.05). However, pregnant women had a 2-fold increase in Pb-P and a 3-fold increase in %Pb-P/Pb-B (both P < 0.01) compared to nonpregnant women. These alterations in Pb concentrations associated with pregnancy are similar to those associated with different ALAD gene variants. We can now better appreciate how pregnancy affects foetal exposure to Pb without the influence of this important genetic factor.

Mercury speciation in whole blood by gas chromatography coupled to ICP-MS with a fast microwave-assisted sample preparation procedure

A simple and fast method is described for simultaneous determination of methylmercury (MeHg), ethylmercury (Et-Hg) and inorganic mercury (Ino-Hg) in blood samples by using capillary gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS) after derivatization and alkaline digestion. Closed-vessel microwave assisted digestion conditions with tetramethylammonium hydroxide (TMAH) have been optimized. Derivatization by using ethylation and propylation procedures have also been evaluated and compared. The absolute detection limits (using a 1 mu L injection) obtained by GC-ICP-MS with ethylation were 40 fg for MeHg and Ino-Hg, respectively, and with propylation were 50, 20 and 50 fg for MeHg, Et-Hg and Ino-Hg, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). Additional validation is provided based on the comparison of results obtained for mercury speciation in blood samples with the proposed procedure and with a previously reported LC-ICP-MS method. With the new proposed procedure no tedious clean-up steps are required and a considerable improvement of the time of analysis was achieved compared to other methods using GC separation.

Influence sexual dimorphism on the persistence of blood parasites in infected Calomys callosus

Gender has long been known to be a contributory factor in the incidence and progression of disorders associated with immune system disregulation. The aims of this experiment were to verify the influences of sexual dimorphism on the persistence of blood parasites out of the acute phase of infection. Male and female Calomys callosus were separated and infected with two strains of Trypanosoma cruzi, and let age until 120 days. Xenogiagnostic, culture of organs and blood, histopathology and lytic antibody percentages were evaluated on late chronic phase. Xenodiagnosis, hemoculture and lytic antibody percentages were positive front 45 until 120 days. For both strains in adrenal and heart, amastigote burdens were present until 45 days, scarcely found on 60 days and absent on 120 days. Steroid hormones, although having a protective role, does not enable animals to get completely rid of the infection. Even without showing apparent signs of pathological unbalance, parasite persists, hidden throughout the host`s body. (C) 2008 Elsevier Ltd. All rights reserved.