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.

Involvement of an Alternative Oxidase in Oxidative Stress and Mycelium-to-Yeast Differentiation in Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.

Increase of core temperature induced by corticotropin-releasing factor and urocortin: A comparative study

This study compared the ability of CRF and UCN1 to induce a thermoregulatory response when centrally injected into rats. The effects of antipyretic drugs and CRF receptor antagonists (CRF(1) and CRF(2)) on the temperature (T) changes induced by these peptides were also investigated. Rectal (rT) and tail skin (T(sk)) temperatures were measured with a thermistor probe while body (bT) temperature was measured with a battery-operated biotelemetry transmitter in male Wistar rats (200 g) every 30 min over a period of 6 h, after intracerebroventricular (i.c.v.) injection of 1 nmol of either CRF or UCN1. Rats were pre-treated with indomethacin (2 mg kg(-1), i.p.) or celecoxib (5 mg kg(-1), p.o.), dexamethasone (0.5 mg kg(-1), s.c.), astressin (a CRF(1)/CRF(2) antagonist, 7 nmol, icy.) or antalarmin (a CRF(1) antagonist, 20 mg kg 1, i.p.). The increase in body temperature induced by CRF was accompanied by a reduction in T(sk) while the response induced by UCN1 was accompanied by an elevation in T(sk). Indomethacin or celecoxib did not change the increases in rT caused by either CRF or UCN1. Although dexamethasone attenuated the increase in rectal temperature in response to CRF, dexamethasone did not modify the response induced by UCN1. Astressin blocked the UCN1-induced hyperthermia and reduced CRF-induced fever. Antalarmin did not modify the hyperthermia in response to UCN1, but reduced the fever evoked by CRF. This study demonstrated that CRF by acting on the CRF(1) receptor induces a prostaglandin-independent fever which seems to depend, at least in part, on the synthesis of other mediators while UCN1 acts on the CRF(2) receptor, promoting a hyperthermic response which seems to be independent on synthesis/release of any mediator. (C) 2010 Elsevier B.V. All rights reserved.

Uncoupling and oxidative stress in liver mitochondria isolated from rats with acute iron overload

One hypothesis for the etiology of cell damage arising from iron overload is that its excess selectively affects mitochondria. Here we tested the effects of acute iron overload on liver mitochondria isolated from rats subjected to a single dose of i.p. 500 mg/kg iron-dextran. The treatment increased the levels of iron in mitochondria (from 21 +/- A 4 to 130 +/- A 7 nmol/mg protein) and caused both lipid peroxidation and glutathione oxidation. The mitochondria of iron-treated rats showed lower respiratory control ratio in association with higher resting respiration. The mitochondrial uncoupling elicited by iron-treatment did not affect the phosphorylation efficiency or the ATP levels, suggesting that uncoupling is a mitochondrial protective mechanism against acute iron overload. Therefore, the reactive oxygen species (ROS)/H(+) leak couple, functioning as a mitochondrial redox homeostatic mechanism could play a protective role in the acutely iron-loaded mitochondria.

Stability Testing of Spray- and Spouted Bed-Dried Extracts of Passiflora alata

The aim of this research was to perform a stability testing of spray- and spouted bed-dried extracts of Passiflora alata Dryander (Passion flower) under stress storage conditions. Spouted bed- and spray-dried extracts were characterized by determination of the average particle diameter (dP), apparent moisture content (XP), total flavonoid content (TF), and vitexin content. Smaller and more irregular particles were generated by the spouted bed system due to a higher attrition rate (surface erosion) inside the dryer. The SB dryer resulted in an end product with higher concentration of flavonoids (approximate to 10%) and lower moisture content (1.6%, dry basis) than the spray dryer, even with both dryers working at similar inlet drying air temperature and ratio between the extract feed flow rate to drying air flow rate (Ws/Wg). Samples of the spouted bed- and spray-dried extracts were stored at two different temperatures (34 and 45 degrees C) and two different relative humidities (52 and 63% RH for 34 degrees C; 52 and 60% RH for 45 degrees C) in order to perform the stability testing. The dried extracts were stored for 28 days and were analyzed every 4 days. The flavonoid vitexin served as the marker compound, which was assayed during the storage period. Results revealed shelf lives ranging from 9 to 184 days, depending on the drying process and storage conditions.

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.

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.

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.

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.

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.

Visible light during mycelial growth and conidiation of Metarhizium robertsii produces conidia with increased stress tolerance

Light conditions during mycelial growth are known to influence fungi in many ways. The effect of visible-light exposure during mycelial growth was investigated on conidial tolerance to UVB irradiation and wet heat of Metarhizium robertsii, an insect-pathogenic fungus. Two nutrient media and two light regimens were compared. Conidia were produced on (A) potato dextrose agar plus yeast extract medium (PDAY) (A1) under dark conditions or (A2) under continuous visible light (provided by two fluorescent lamps with intensity 5.4 W m-2). For comparison, the fungus was also produced on (B) minimal medium (MM) under continuous-dark incubation, which is known to produce conidia with increased tolerance to heat and UVB radiation. The UVB tolerances of conidia produced on PDAY under continuous visible light were twofold higher than conidia produced on PDAY medium under dark conditions, and this elevated UVB tolerance was similar to that of conidia produced on MM in the dark. The heat tolerance of conidia produced under continuous light was, however, similar to that of conidia produced on MM or PDAY in the dark. Conidial yield on PDAY medium was equivalent when the fungus was grown either under continuous-dark or under continuous-light conditions.

Infectivity of Strongyloides venezuelensis is influenced by variations in temperature and time of culture

The present research investigated the influence of temperature and time of larvae culture on the infectivity of Strongyloides venezuelensis. Mice were infected s.c. with 1500 larvae of S. venezuelensis maintained at 28 degrees C for three days of culture (dc), 28 degrees C for seven dc or 18 degrees C for seven dc. On days 1,3, 5, 7, 14 and 21 post-infection the animals were sacrificed and cell numbers in the blood, peritoneal cavity fluid (PCF), broncoalveolar fluid (BALF), cytokines, immunoglobulins, number of parasites and eggs/g of feces were quantified. Results demonstrated an increase in eosinophils and mononuclear cells in the blood, PCF and HALF of infected mice. Larvae at 28 degrees C/3dc induced earlier eosinophils in the PCF and HALF as opposed to larvae at 28 degrees C/7dc and 18 degrees C/7dc. Larvae at 28 degrees C/7dc induced higher synthesis of IL-4. IL-5 and IL-10 on days Sand 7 post-infection. Larvae at 28 degrees C/3dc in culture induced higher synthesis of IL-12 than larvae of seven dc, but time in culture induced better synthesis of IFN-gamma, after larval migration had ceased and only adult worms were present. Larvae at 28 degrees C/3dc in culture induced higher synthesis of IgG and IgG1 and expelled less female parasites than larvae cultivated for seven days. In conclusion, it was observed that the infectivity of S. venezuelensis is influenced by variations in temperature and time of culture. (C) 2010 Elsevier Inc. All rights reserved.

Fungal tyrosine betaine, a novel secondary metabolite from conidia of entomopathogenic Metarhizium spp. fungi

Fungi, including the entomopathogenic deuteromycete Metarhizium anisopliae, produce a wide diversity of secondary metabolites that either can be secreted or stored in specific developmental structures, e.g., conidia. Some secondary metabolites, such as pigments, polyols and mycosporines, are associated with pathogenicity and/or fungal tolerance to several stress-inducing environmental factors, including temperature and solar radiation extremes. Extracts of M. anisopliae var. anisopliae (strain ESALQ-1037) conidia were purified by chromatographic procedures and the isolated compounds analyzed by (1)H and (13)C nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. LC-MS analyses were carried out to search for mycosporines (the initial targets), but no compounds of this class were detected. A molecule whose natural occurrence was previously undescribed was identified. It consists of betaine conjugated with tyrosine, and the structure was identified as 2-([1-carboxy-2-(4-hydroxyphenyl)ethyl]amino)-N,N,N-trimethyl-2-oxoethanammonium. mannitol was the predominant compound in the alcoholic conidial extract, but no amino acids other than tyrosine were found to be conjugated with betaine in conidia. The fungal tyrosine betaine was detected also in conidial extracts of three other M. anisopliae var. anisopliae (ARSEF 1095, 5626 and 5749) and three M. anisopliae var. acridum isolates (ARSEF 324, 3391 and 7486), but it was not detected in Aspergillus nidulans conidial extract (ATCC 10074). (C) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Association Among Microalbuminuria and Oxidative Stress Biomarkers in Patients With Type 2 Diabetes

Aim: Hyperglycemia in diabetes mellitus (DM) may be one of the most important factors responsible for the development of oxidative stress, which promotes the main complications in DM patients. Therefore, this study evaluated if the hyperglycemia could be related to oxidative stress biomarkers, lipid profile, and renal function in type 2 diabetes patients without clinic complications. Methods: Plasmatic malondialdehyde (MDA), serum protein carbonyl (PCO), serum creatinine levels, microalbuminuria, glycated hemoglobin, and lipid profile were analyzed in 37 type 2 diabetic patients and 25 subjects with no diabetes. Results: Serum creatinine levels were within the reference values, but microalbuminuria presented increased levels in all the patients compared with controls (P G 0.05) and above of the reference values. The MDA, PCO, low- density lipoprotein, and triglyceride levels showed positive correlation with microalbuminuria levels. Moreover, glycated hemoglobin presented positive correlation with MDA, PCO, and microalbuminuria levels. Conclusions: The hyperglycemia could be responsible for the increase of the microalbuminuria levels and for the oxidation process in lipids and proteins in DM patients. Therefore, we suggested that the microvascular lesion is a direct consequence from hyperglycemia and an indirect one from the increased oxidative stress. Malondialdehyde and protein carbonyl levels could be suggested as additional biochemical evaluation to verify tissue damage in type 2 DM patients.