Penetration of Quantum Dot Particles Through Human Skin

The skin is a large and accessible area of the body, offering the possibility to be used as an alternative route for drug delivery. In the last few years strong progress has been made on the developing of nanoparticulate systems for specific applications. The interaction of such small particles with human skin and their possible penetration attracted some interest from toxicological as well as from drug delivery perspectives. As size is assumed to play a key role, the aim of the present work was to investigate the penetration profile of very small model particles (similar to 4 nm) into excised human skin under conditions chosen to mimic the in vivo situation. Possible application procedures such as massaging the formulation (5 to 10 minutes) were analyzed by non-invasive multiphoton- and confocal laser scanning microscopy (MPM, CLSM). Furthermore, the application on damaged skin was taken into account by deliberately removing parts of the stratum corneum. Although it was clearly observed that the mechanical actions affected the distribution pattern of the QDs on the skin surface, there was no evidence of penetration into the skin in all cases tested. QDs could be found in deeper layers only after massaging of damaged skin for 10 min. Taking these data into account, obtained on the gold standard human skin, the potential applications of nanoparticulate systems to act as carrier delivering drugs into intact skin might be limited and are only of interest for partly damaged skin.

Rheological, mechanical and mucoadhesive properties of thermoresponsive, bioadhesive binary mixtures composed of poloxamer 407 and carbopol 974P designed as platforms for implantable drug delivery systems for use in the oral cavity

This study described the formulation and characterisation of the viscoelastic, mechanical and mucoadhesive properties of thermoresponsive, binary polymeric systems composed of poloxamer (P407) and poly(acrylic acid, C974P) that were designed for use as a drug delivery platform within the oral cavity. Monopolymeric and binary polymeric formulations were prepared containing 10, 15 and 20% (w/w) poloxamer (407) and 0.10-0.25% (w/w) poly(acrylic acid, 934P). The flow theological and viscoelastic properties of the formulations were determined using controlled stress and oscillatory rheometry, respectively, the latter as a function of temperature. The mechanical and mucoadhesive properties (namely the force required to break the bond between the formulation and a pre-hydrated mucin disc) were determined using compression and tensile analysis, respectively. Binary systems composed of 10% (w/w) P407 and C934P were elastoviscous, were easily deformed under stress and did not exhibit mucoadhesion. Formulations containing 15 or 20% (w/w) Pluronic P407 and C934P exhibited a sol-gel temperature T(sol/gel), were viscoelastic and offered high elasticity and resistance to deformation at 37 degrees C. Conversely these formulations were elastoviscous and easily deformed at temperatures below the sol-gel transition temperature. The sol-gel transition temperatures of systems containing 15% (w/w) P407 were unaffected by the presence of C934P; however, increasing the concentration of C934P decreased the T(sol/gel) in formulations containing 20%(w/w) P407. Rheological synergy between P407 and C934P at 37 degrees C was observed and was accredited to secondary interactions between these polymers, in addition to hydrophobic interactions between P407 micelles. Importantly, formulations composed of 20% (w/w) P407 and C934P exhibited pronounced mucoadhesive properties. The ease of administration (below the T(sol/gel)) in conjunction with the viscoelastic (notably high elasticity) and mucoadhesive properties (at body temperature) render the formulations composed of 20% (w/w) P407 and C934P as potentially useful platforms for mucoadhesive, controlled topical drug delivery within the oral cavity. (c) 2009 Published by Elsevier B.V.

Functional characterization of the Aspergillus nidulans methionine sulfoxide reductases (msrA and msrB)

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Here. we describe the homologs of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage. (C) 2009 Elsevier Inc. All rights reserved.

An evaluation, using the comet assay and the micronucleus test, of the antigenotoxic effects of chlorophyll b in mice

We investigated the effects of the dietary pigment chlorophyll b (CLb) on cisplatin (cDDP)-induced oxidative stress and DNA damage, using the comet assay in mouse peripheral blood cells and the micronucleus (MN) test in bone marrow and peripheral blood cells. We also tested for thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) in liver and kidney tissues, as well as catalase (CAT) activity and GSH in total blood. CLb (0.2 and 0.5 mg/kg b.w.) was administrated by gavage every day for 13 days. On the 14th day of the experiment, 6 mg/kg cDDP or saline was delivered intraperitoneally. Treatment with cDDP led to a significant decrease in DNA migration and an increase in MN frequency in both cell types, bone marrow and peripheral blood cells. In the kidneys of mice treated with cDDP, TBARS levels were increased, whereas GSH levels were depleted in kidney and liver. In mice that were pretreated with CLb and then treated with cDDP, TBARS levels maintained normal concentrations and GSH did not differ from cDDP group. The improvement of oxidative stress biomarkers after CLb pre-treatment was associated with a decrease in DNA damage, mainly for the highest dose evaluated. Furthermore, CLb also slightly reduced the frequency of chromosomal breakage and micronucleus formation in mouse bone marrow and peripheral blood cells. These results show that pre-treatment with CLb attenuates cDDP-induced oxidative stress, chromosome instability, and lipid peroxidation. (C) 2011 Elsevier B.V. All rights reserved.

Pre and post-natal exposure to ambient level of air pollution impairs memory of rats: the role of oxidative stress

The aims of this study were to evaluate whether air pollution during pre-natal and post-natal phases change habituation and short-term discriminative memories and if oxidants are involved in this process. As secondary objectives, it was to evaluate if the change of filtered to nonfiltered environment could protect the cortex of rats against oxidative stress as well as to modify the behavior of these animals. Wistar, male rats were divided into four groups (n = 12/group): pre and post-natal exposure until adulthood to filtered air (FA); pre-natal period to nonfiltered air (NFA-FA); until (21st post-natal day) and post-natal to filtered air until adulthood (PND21); prenatal to filtered air until PND21 and post-natal to nonfiltered air until adulthood (FA-NFA); pre and post-natal to nonfiltered air (NFA). After 150 days of air pollution exposure, animals were tested in the spontaneous object recognition test to evaluate short-term discriminative and habituation memories. Rats were euthanized; blood was collected for metal determination; cortex dissected for oxidative stress evaluation. There was a significant increase in malondialdehyde (MDA) levels in the NFA group when compared to other groups (FA: 1.730 +/- 0.217; NFA-FA: 1.101 +/- 0.217; FA-NFA: 1.014 +/- 0.300; NFA: 5.978 +/- 1.920 nmol MDA/mg total proteins; p = 0.007). NFA group presented a significant decrease in short-term discriminative (FA: 0.603 +/- 0.106; NFA-FA: 0.669 +/- 0.0666; FA-NFA: 0.374 +/- 0.178; NFA: -0.00631 +/- 0.106 sec; p = 0.006) and an improvement in habituation memories when compared to other groups. Therefore, exposure to air pollution during both those periods impairs short-term discriminative memory and cortical oxidative stress may mediate this process.

Mercury exposure and oxidative stress in communities of the Brazilian Amazon

This study was designed to assess possible associations between biomarkers of mercury (Hg) exposure and oxidative stress in fish-eating Amazonian communities. Clinical samples were obtained from riparians living in the Brazilian Amazon. Biomarkers of oxidative stress (glutathione - GSH, glutathione peroxidase - GSH-Px, catalase - CAT, activity and reactivation index of delta-aminolevulinate dehydratase - ALA-D (R%) were determined in blood. Total Hg was measured in whole blood (B-Hg), plasma (P-Hg) and hair (H-Hg). Association between biomarkers of Hg exposure and oxidative stress were examined using multiple regression models, including age, gender, alcohol consumption, smoking status, fish consumption and then stratified for gender. Significant inverse relations were observed between GSH-Px, GSH, CAT, ALA-D activity and B-Hg or H-Hg (p<0.05). ALA-D reactivation index was positively related to B-Hg (p<0.0001). P-Hg was directly related to ALA-D reactivation index and inversely associated with GSH-Px, GSH, and ALA-D activity (p<0.05). When stratified for gender, women showed significant inverse associations between all biomarkers of Hg exposure and CAT (p<0.05) or GSH (p<0.05), while for men only P-Hg showed a significant inverse relation with GSH (p<0.001). Our results clearly demonstrated an association between Hg exposure and oxidative stress. Moreover, for B-Hg, P-Hg and H-Hg gender differences were present. (C) 2009 Elsevier B.V. All rights reserved.

Mercury Exposure Increases Circulating Net Matrix Metalloproteinase (MMP)-2 and MMP-9 Activities

Mercury (Hg) exposure causes health problems that may result from increased oxidative stress and matrix metalloproteinase (MMP) levels. We investigated whether there is an association between the circulating levels of MMP-2, MMP-9, their endogenous inhibitors (the tissue inhibitors of metalloproteinases; TIMPs) and the circulating Hg levels in 159 subjects environmentally exposed to Hg. Blood and plasma Hg were determined by inductively coupled plasma-mass spectrometry (ICP-MS). MMP and TIMP concentrations were measured in plasma samples by gelatin zymography and ELISA respectively. Thiobarbituric acid-reactive species (TBARS) were measured in plasma to assess oxidative stress. Selenium (Se) levels were determined by ICP-MS because it is an antioxidant. The relations between bioindicators of Hg and the metalloproteinases levels were examined using multivariate regression models. While we found no relation between blood or plasma Hg and MMP-9, plasma Hg levels were negatively associated with TIMP-1 and TIMP-2 levels, and thereby with increasing MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios, thus indicating a positive association between plasma Hg and circulating net MMP-9 and MMP-2 activities. These findings provide a new insight into the possible biological mechanisms of Hg toxicity, particularly in cardiovascular diseases.

Protective properties of quercetin against DNA damage and oxidative stress induced by methylmercury in rats

Aim of the study was to find out whether consumption of quercetin (QC), an abundant flavonoid in the human diet, protects against DNA damage caused by exposure to organic mercury. Therefore, rats were treated orally with methylmercury (MeHg) and the flavonoid with doses that reflect the human exposure. The animals received MeHg (30 mu g/kg/bw/day), QC (0.5-50 mg/kg/bw/day), or combinations of both over 45 days. Subsequently, the glutathione levels (GSH) and the activities of glutathione peroxidase (GPx) and catalase (CAT) were determined, and DNA damage was measured in hepatocytes and peripheral leukocytes in single cell gel electrophoresis assays. MeHg decreased the concentration of GSH and the activity of GPx by 17 and 12%, respectively and caused DNA damage to liver and blood cells, while with QC no such effects were seen. When the flavonoid was given in combination with MeHg, the intermediate and the highest concentrations (5.0 and 50.0 mg/kg/bw/day) were found to cause DNA protection; DNA migration was reduced by 54 and 65% in the hepatocytes and by 27 and 36% in the leukocytes; furthermore, the reduction in GSH and GPx levels caused by MeHg treatment was restored. In summary, our results indicate that consumption of QC-rich foods may protect Hg-exposed humans against the adverse health effects of the metal.

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.

Synthesis, antichagasic in vitro evaluation, cytotoxicity assays, molecular modeling and SAR/QSAR studies of a 2-phenyl-3-(1-phenyl-1H-pyrazol-4-yl)-acrylic acid benzylidene-carbohydrazide series

Chagas disease (American trypanosomiasis) is one of the most important parasitic diseases with serious social and economic impacts mainly on Latin America. This work reports the synthesis, in vitro trypanocidal evaluation, cytotoxicity assays, and molecular modeling and SAR/QSAR studies of a new series of N-phenylpyrazole benzylidene-carbohydrazides. The results pointed 6k (X = H, Y = p-NO(2), pIC(50) = 4.55 M) and 6l (X = F, Y = p-CN, pIC(50) = 4.27 M) as the most potent derivatives compared to crystal violet (pIC(50) = 3.77 M). The halogen-benzylidene-carbohydrazide presented the lowest potency whereas 6l showed the most promising pro. le with low toxicity (0% of cell death). The best equation from the 4D-QSAR analysis (Model 1) was able to explain 85% of the activity variability. The QSAR graphical representation revealed that bulky X-substituents decreased the potency whereas hydrophobic and hydrogen bond acceptor Y-substituents increased it. (C) 2008 Elsevier Ltd. All rights reserved.

Effects of repetitive stress during the acute phase of Trypanosoma cruzi infection on chronic Chagas` disease in rats

The effect of repetitive stress during acute infection with Trypanosoma cruzi (T. cruzi) on the chronic phase of ensuing Chagas` disease was the focus of this investigation. The aim of this study was to evaluate in Wistar rats the influence of repetitive stress during the acute phase of infection (7 days) with the Y strain of T. cruzi on the chronic phase of the infection (at 180 days). Exposure to ether vapor for 1min twice a day was used as a stressor. Repetitive stress enhanced the number of circulating parasites and cardiac tissue disorganization, from a moderate to a severe diffuse mononuclear inflammatory process and the presence of amastigote burden in the cardiac fibers. Immunological parameters revealed that repetitive stress triggered a reduced concanavalin A induced splenocyte proliferation in vitro with major effects on the late chronic phase. Serum interleukin-12 concentration decreased in both stressed and infected rats in the early phase of infection although it was higher on 180 days post-infection. These results suggest that repetitive stress can markedly impair the host`s immune system and enhance the pathological process during the chronic phase of Chagas` disease.

Evidence of caspase-mediated apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom

The aim of this work was to investigate the involvement of caspases in apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAA0. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in ""Asp and Glu"" residues. It displays high specificity toward hydrophobic L-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H(2)O(2) production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances. Published by Elsevier Inc.

Antiviral and antiparasite properties of an L-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence (Retracted article. See vol. 80, pg. 288, 2010)

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pl of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs. (C) 2008 Elsevier Inc. All rights reserved.

Trypanosoma cruzi: Effects of social stress in Calomys callosus a natural reservoir of infection

Social environment can represent a major source of stress affecting cortisol and/or corticosterone levels, thereby altering the immune response. We have investigated the effects of social isolation on the development of Trypanosoma cruzi infection in female Calomys callosus, a natural reservoir of this protozoan parasite. Animals were divided in groups of five animals each. The animals of one group were kept together in a single cage. In a second group, four females were kept together in a cage with one male. In the final group, five individuals were kept isolated in private cages. The isolated animals showed body weight reduction, decreased numbers of peritoneal macrophages, lower global leucocytes counts, smaller lytic antibody percentage and a significantly higher level of blood parasites compared to the other animals. Their behavior was also altered. They were more aggressive than grouped females, or females exposed to the presence of a male. These results suggest that isolation creates a distinct social behavior in which immunity is impaired and pathogenesis is enhanced. (C) 2008 Elsevier Inc. All rights reserved.

The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats

Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diet`s effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diet`s effects on genomic stability and DNA methylation. (C) 2011 Elsevier ay. All rights reserved.