Diazinon toxicity in hepatic and spleen mononuclear cells is associated to early induction of oxidative stress.

Diazinon is an organophosphorus pesticide, which may have potential toxic effects on the liver and immune system; however, the underlying mechanisms remain mostly unidentified. This work is aimed at evaluating the oxidative stress and cell cycle alterations elicited by low-dose diazinon in a rat liver cell line (BRL-3A) and spleen mononuclear cells (SMC) from Wistar rats. Diazinon (10-50 µM) caused early reactive oxygen species (ROS) generation (from 4 h) as well as increased O2•- level (from 0.5 h), which led to subsequent lipid peroxidation at 24 h, in BRL-3A cells. In SMC, diazinon (20 µM) produced similar increases in ROS levels, at 4 and 24 h, with the highest O2•- level being found at 4 h. Low-dose diazinon induced G1-phase arrest and cell death in hepatic cells and SMC. Therefore, diazinon could affect the liver and the immunological system through the premature oxidative stress induction.Abbreviations: O2•-: superoxide anion radical; ROS: reactive oxygen species; SMC: spleen mononuclear cells; TBARS: thiobarbituric acid reactive substances.

Where Does the Peanut Smut Pathogen, Thecaphora frezii, Fit in the Spectrum of Smut Diseases?

Smut fungi, such as Ustilago maydis, have been studied extensively as a model for plant-pathogenic basidiomycetes. However, little attention has been paid to smut diseases of agronomic importance that are caused by species of the genus Thecaphora, probably due to their more localized distribution. Peanut smut incited by Thecaphora frezii has been reported only in South America, and Argentina is the only country where this disease has been noted in commercial peanut production. In this work, important advances in deciphering T. frezii specific biology/pathobiology in relation to potato (T. solani), wheat (U. tritici), and barley (U. nuda) smuts are presented. We summarize the state of knowledge of fungal effectors, functionally characterized to date in U. maydis and most recently in T. thlaspeos, as well as the potential to be present in other Thecaphora species involved in dicot-host interactions like T. frezii-peanut. We also discuss applicability and limitations of currently available methods for identification of smut fungi in different situations and management strategies to reduce their impact on agri-food quality. We conclude by describing some of the challenges in elucidating T. frezii strategies that allow it to infect the host and tolerate or evade plant immune defense mechanisms, and assessing other aspects related to pest control and their implications for human health.

Bioaccessibility of polyphenols and antioxidant properties of the white grape by simulated digestion and Caco-2 cell assays: Comparative study with its winemaking product.

The primary objective of this study was to assess the changes on phenolic composition and AC (antioxidant capacity) of white grape and its winemaking product, during in vitro gastrointestinal (GI) digestion. Phenolic compounds were evaluated by HPLC-MS/MS. The AC was measured by in vitro (FRAP, ABTS and DPPH) and cellular (Caco-2 cells) assays. Digestion had a reducing effect on phenolic content, being only 31% and 67% of native polyphenols from grapes and wines, respectively, potentially bioaccessible. At same polyphenol concentration, cellular AC of nondigested and digested foods was the same, indicating that changes in phenolic profile did not modify the bioactivity. Phenolic acids, in addition to quercetin, were the most resistant polyphenols to digestion, and would be the most relevant to explain the biological activity of digested foods. Results indicate that the changes occurred in the native phenolic profile of foods as a consequence of GI digestion, do not modify the bioactivity of white grapes and wines.

Biosynthesized silver nanoparticles: Decoding their mechanism of action in Staphylococcus aureus and Escherichia coli.

The oxidative stress generation in bacteria by the presence of antibiotics (in this case silver nanoparticles (AgNPs)) is already widely known. Previously, we demonstrated that AgNPs generate oxidative stress in Staphylococcus aureus and Escherichia coli mediated by the increase of reactive oxygen species (ROS). In this work we are demonstrating the consequences of the oxidative stress by the presence of AgNPs; these bacterial strains increased the levels of oxidized proteins and lipids. In addition, it was possible to determine which reactive oxygen species are mainly responsible for the oxidative damage to macromolecules. Also, we found that the bacterial DNA was fragmented and the membrane potential was modified. This increase in the levels of ROS found in both, S. aureus and E. coli, was associated with the oxidation of different types of important macromolecules for the normal functioning of cell, so the oxidative stress would be one of the mechanisms by which the AgNPs would exert their toxicity in both strains, one Gram positive and the other Gram negative of great clinical relevance.

IL-17-Mediated Immunity Controls Skin Infection and T Helper 1 Response during Experimental Microsporum canis Dermatophytosis.

Despite worldwide prevalence of superficial mycoses, the immune response in dermatophytosis has scarcely been investigated. In this study, we developed a model of superficial skin infection in C57BL/6 mice with Microsporum canis, a highly prevalent human pathogen. This model mimics mild inflammatory human dermatophytosis, characterized by neutrophil recruitment and fungal invasion limited to the epidermis and exhibits the establishment of a specific T helper type 17 immune response during infection. By using IL-17RA- or IL-17A/F-deficient mice we showed that, in the absence of a functional IL-17 pathway, M. canis extensively colonizes the epidermis and promotes an exaggerated skin inflammation and a shift to an IFN-γ-mediated (T helper type 1) response. IL-17 signaling was not involved in neutrophil influx to skin or fungal invasion to deeper tissues. Finally, this study shows that skin langerin-expressing cells contribute to the antifungal T helper type 17 response in vivo. In conclusion, these data directly show a dual function of IL-17 cytokines in dermatophytosis by controlling superficial infection and down-modulating a T helper type 1 antifungal response.

3-Hydroxykynurenine, a Tryptophan Metabolite Generated during the Infection, Is Active Against Trypanosoma cruzi.

The antiparasitic activity of 3-hydroxykynurenine (3-HK), one of the major tryptophan catabolites of the kynurenine pathway, against both Trypanosoma cruzi evolutive forms that are important for human infection, trypomastigotes (Tps) and amastigotes (Am), possible targets in the parasite and the drug toxicity to mammalian cells have been investigated. 3-HK showed a potent activity against Am with IC50 values in the micromolar concentration range, while the IC50 values to cause Tps death was ∼6000-times higher, indicating that the replicative form present in the vertebrate hosts is much more susceptible to 3-HK than bloodstream Tps. In addition, 3-HK showed activity against Tps and Am, at concentrations that did not exhibit toxicity to mammalian cells. Ultrastructural analysis and flow cytometry studies indicated that Am and Tps mitochondrion and nuclei contain 3-HK targets. The potency and selectivity of 3-HK, which is generated during T. cruzi infection in human and mice, suggest that 3-HK may be a suitable candidate for drug research and development for Chagas disease.

The aflatoxin B1 -fumonisin B1 toxicity in BRL-3A hepatocytes is associated to induction of cytochrome P450 activity and arachidonic acid metabolism.

Human oral exposure to aflatoxin B1 (AFB1 ) and fumonisin B1 (FB1 ) is associated with increased hepatocellular carcinoma. Although evidence suggested interactive AFB1 -FB1 hepatotoxicity, the underlying mechanisms remain mostly unidentified. This work was aimed at evaluating the possible AFB1 -FB1 interplay to induce genetic and cell cycle toxicities in BRL-3A rat hepatocytes, reactive oxygen species (ROS) involvement, and the AFB1 metabolizing pathways cytochrome P450 (CYP) and arachidonic acid (ArAc) metabolism as ROS contributors. Flow cytometry of stained BRL-3A hepatocytes was used to study the cell cycle (propidium iodide), ROS intracellular production (DCFH-DA, HE, DAF-2 DA), and phospholipase A activity (staining with bis-BODIPY FL C11-PC). The CYP1A activity was assessed by the 7-ethoxyresorufin-O-deethylase (EROD) assay. Despite a 48-h exposure to FB1 (30 µM) not being genotoxic, the AFB1 (20 µM)-induced micronucleus frequency was overcome by the AFB1 -FB1 mixture (MIX), presumably showing toxin interaction. The mycotoxins blocked G1/S-phase, but only MIX caused cell death. Overall, the oxidative stress led these alterations as the pretreatment with N-acetyl-l-cysteine reduced such toxic effects. While AFB1 had a major input to the MIX pro-oxidant activity, with CYP and ArAc metabolism being ROS contributors, these pathways were not involved in the FB1 -elicited weak oxidative stress. The MIX-induced micronucleus frequency in N-acetyl-l-cysteine pretreated cells was greater than that caused by AFB1 without antioxidants, suggesting enhanced AFB1 direct genotoxicity probably owing to the higher CYP activity and ArAc metabolism found in MIX. The metabolic pathways modulation by AFB1 -FB1 mixtures could raise its hepatocarcinogenic properties.

IL-6 promotes M2 macrophage polarization by modulating purinergic signaling and regulates the lethal release of nitric oxide during Trypanosoma cruzi infection.

The production of nitric oxide (NO) is a key defense mechanism against intracellular pathogens but it must be tightly controlled in order to avoid excessive detrimental oxidative stress. In this study we described a novel mechanism through which interleukin (IL)-6 mediates the regulation of NO release induced in response to Trypanosoma cruzi infection. Using a murine model of Chagas disease, we found that, in contrast to C57BL/6 wild type (WT) mice, IL-6-deficient (IL6KO) mice exhibited a dramatic increase in plasma NO levels concomitant with a significantly higher amount of circulating IL-1ß and inflammatory monocytes. Studies on mouse macrophages and human monocytes, revealed that IL-6 decreased LPS-induced NO production but this effect was abrogated in the presence of anti-IL-1ß and in macrophages deficient in the NLRP3 inflammasome. In accordance, while infected WT myocardium exhibited an early shift from microbicidal/M1 to anti-inflammatory/M2 macrophage phenotype, IL6KO cardiac tissue never displayed a dominant M2 macrophage profile that correlated with decreased expression of ATP metabolic machinery and a lower cardiac parasite burden. The deleterious effects of high NO production-induced oxidative stress were evidenced by enhanced cardiac malondialdehyde levels, myocardial cell death and mortality. The survival rate was improved by the treatment of IL-6-deficient mice with a NO production-specific inhibitor. Our data revealed that IL-6 regulates the excessive release of NO through IL-1ß inhibition and determines the establishment of an M2 macrophage profile within infected heart tissue.

Toxin distribution and sphingoid base imbalances in Fusarium verticillioides-infected and fumonisin B1-watered maize seedlings.

Fusarium verticillioides is a major maize pathogen and there are susceptible and resistant cultivars to this fungal infection. Recent studies suggest that its main mycotoxin fumonisin B1 (FB1) may be involved in phytopathogenicity, but the underlying mechanisms are mostly still unknown. This work was aimed at assessing whether FB1 disseminates inside the plants, as well as identifying possible correlations between the maize resistant/susceptible phenotype and the unbalances of the FB1-structurally-related sphingoid base sphinganine (Sa) and phytosphingosine (Pso) due to toxin accumulation. Resistant (RH) and susceptible hybrid (SH) maize seedlings grown from seeds inoculated with a FB1-producer F. verticillioides and from uninoculated ones irrigated with FB1 (20 ppm), were harvested at 7, 14 and 21 days after planting (dap), and the FB1, Sa and Pso levels were quantified in roots and aerial parts. The toxin was detected in roots and aerial parts for inoculated and FB1-irrigated plants of both hybrids. However, FB1 levels were overall higher in SH seedlings regardless of the treatment (infection or watering). Sa levels increased substantially in RH lines, peaking at 54-fold in infected roots at 14 dap. In contrast, the main change observed in SH seedlings was an increase of Pso in infected roots at 7 dap. Here, it was found that FB1 disseminates inside seedlings in the absence of FB1-producer fungal infections, perhaps indicating this might condition the fungus-plant interaction before the first contact. Furthermore, the results strongly suggest the existence of at least two ceramide synthase isoforms in maize with different substrate specificities, whose differential expression after FB1 exposure could be closely related to the susceptibility/resistance to F. verticillioides.

Effect of surface charge on the interfacial orientation and conformation of FB1 in model membranes.

The toxicity of FB1 is usually explained through the enzymatic disruption of lipidic metabolism. However, it may lie in the thermodynamics of the membrane and its cooperative phase behavior rather than in the activity of individual proteins. Here, we investigate the effects of FB1 at the molecular and mesoscopic levels in FB1-phospholipid mixed Langmuir films. Mean molecular area vs FB1 molar fractions (x(FB1)) and phase diagram analysis allowed us to define miscibility conditions and phase states at different x(FB1). Surface potential measurements, evaluated as a function of the molecular packing and x(FB1), revealed the FB1-induced change in the collective dipolar reorientation leaded to neutralization of charged films. Size, shape, and distribution of 2D-domain analysis from epifluorescence data suggested the increase in the mixing entropy and film relaxation rate. Finally, PM-IRRAS revealed the orientation of FB1 with the amine end (zwitterionic and negatively charged monolayers) or the tricarballylic acid end (positively charged monolayers) pointing to the air. The globular-extended conformational equilibrium of FB1 is dynamically defined by the membrane charge becoming a toxicity enhancing factor. The specificity of the toxin-protein interaction might also be perturbed by the FB1-induced remodeling of the membrane topography by affecting the raft-like platforms where membrane enzymes are considered to be located.

Reactive oxygen species sources and biomolecular oxidative damage induced by aflatoxin B1 and fumonisin B1 in rat spleen mononuclear cells.

Aflatoxin B1 (AFB(1)) and fumonisin B1 (FB(1)) are mycotoxins widely found as cereal contaminants. Their immunotoxicities predispose to infectious diseases and may alter the tumor immunosurveillance of human and animals, but the mechanisms underlying have not been fully elucidated, and the induction of oxidative stress has been proposed as a probable mechanism. This work was aimed at evaluating in spleen mononuclear cells (SMC) from Wistar rats the effects of the exposure, in vitro for up to 48 h, to 20 µM AFB(1), 10 µM FB(1) and AFB(1)-FB(1) mixture (MIX), over cellular oxidative status, as well as at elucidating the contribution of different reactive oxygen species (ROS) to biomolecular oxidative damage, the biochemical pathways involved, and the probable interaction of both toxins to induce oxidative stress. All the treatments increased total ROS and oxidation of biomolecules, with MIX having the greatest effects. However, only MIX increased superoxide anion radical. The main ROS involved in oxidation of proteins, lipids and DNA appear to be hydrogen peroxide and hydroxyl radical. The mitochondrial complex I and CYP450 were involved in the ROS generation induced by all treatments. The NADPH oxidase system was induced by FB1 and MIX. The arachidonic acid metabolism contributed to the ROS formation induced by AFB(1) and MIX. These results demonstrate that an interaction between AFB(1) and FB(1) occur in the oxidative stress induction, and show the biochemical pathways involved in ROS generation in SMC. The oxidative stress could mediate the AFB(1) and FB(1) individual and combined immunotoxicities.

Fumonisins: probable role as effectors in the complex interaction of susceptible and resistant maize hybrids and Fusarium verticillioides.

Fusarium verticillioides is best known for its worldwide occurrence on maize resulting in highly variable disease symptoms, ranging from asymptomatic to severe rotting and wilting and fumonisin production. The aim of this study was to investigate the influence of hybrid genotypes in the early stages of F. verticillioides infection, and the role of fumonisins as effectors in the outcome of this complex interaction. Disease symptoms, growth parameters, root morphology, and fungal colonization were evaluated at 7, 14, and 21 days after planting in seedlings from maize seeds of resistant (RH) and susceptible (SH) hybrids inoculated with F. verticillioides or watered with solutions of fumonisins. F. verticillioides induced growth enhancement or retardation depending on the plant genetic background and the fungal colonization rate, while fumonisins caused severe reduction in biomass and fitness. Seedlings watered with high fumonisin concentrations displayed lesions similar to those seen in F. verticillioides maize seedling disease, and also elicited inhibitory effects on root growth and morphology and on functional properties. In summary, these data strongly suggest a dual role for fumonisins in the F. verticillioides-maize interaction, acting as pathogenic factors at high concentrations, or triggering the plant detoxification mechanisms at low levels.

The lipid-mediated hypothesis of fumonisin B1 toxicodynamics tested in model membranes.

The disruption of lipidic metabolism was considered a good candidate to explain FB1 toxicity mechanism. In the present work we investigated molecular organizational changes induced by FB1-biomembrane interaction possibly involved in mycotoxic effects. FB1 was self-aggregated with a critical micellar concentration of 1.97 mM. FB1 (0-81.4 microM), decreased in a dose-dependent manner, the fluorescence anisotropy of TMA-DPH (from 0.349+/-0.003 to 0.1720+/-0.0035) in dpPC bilayers, whilst no differences were registered with DPH. At 5.6 microM in the subphase, FB1 increased the lateral surface pressure (pi) of a Langmuir film to an extent that depended on the monolayer composition (Deltapi dpPC:DOTAP 3:1>Deltapi dpPC:dpPA3:1>Deltapi dpPC), the molecular packing (Deltapi decreased linearly as a function of the initial pi) and the subphase pH (Deltapi pH 2.6>Deltapi pH 7.4 and maximal pi allowing the drug penetration pi cut-off was 34.3 and 27.7 mN/m at pH 2.63 and 7.4, respectively). FB1 increased the surface potential of dpPC and dpPC:DOTAP monolayers and decreased that of dpPC:dpPA. This suggested that FB1 acquired different orientations and/or foldings depending on the surface electrostatics and the toxin charge state. Moreover, FB1-lipid interactions were transduced into long-range effects at the mesoscopic level affecting the lipidic self-separated lateral domains shape and density.