Tl(I) and Tl(III)-induce genotoxicity, reticulum stress and autophagy in PC12 Adh cells.

Thallium (Tl) and its two cationic species, Tl(I) and Tl(III), are toxic for most living beings. In this work, we investigated the effects of Tl (10-100 µM) on the viability and proliferation capacity of the adherent variant of PC12 cells (PC12 Adh cells). While both Tl(I) and Tl(III) halted cell proliferation from 24 h of incubation, their viability was ~ 90% even after 72 h of treatment. At 24 h, increased levels of γH2AX indicated the presence of DNA double-strand breaks. Simultaneously, increased expression of p53 and its phosphorylation at Ser15 were observed, which were associated with decreased levels of p-AKTSer473 and p-mTORSer2448. At 72 h, the presence of large cytoplasmic vacuoles together with increased autophagy predictor values suggested that Tl may induce autophagy in these cells. This hypothesis was corroborated by images obtained by transmission electron microscopy (TEM) and from the decreased expression at 72 h of incubation of SQSTM-1 and increased LC3ß-II to LC3ß-I ratio. TEM images also showed enlarged ER that, together with the increased expression of IRE1-α from 48 h of incubation, indicated that Tl-induced ER stress preceded autophagy. The inhibition of autophagy flux with chloroquine increased cell mortality, suggesting that autophagy played a cytoprotective role in Tl toxicity in these cells. Together, results indicate that Tl(I) or Tl(III) are genotoxic to PC12 Adh cells which respond to the cations inducing ER stress and cytoprotective autophagy.

Tl(I) and Tl(III) induce reticulum stress in MDCK cells.

The effects of the exposure of proliferating MDCK cells to thallium [Tl(I) or Tl(III)] on cell viability and proliferation were investigated. Although Tl stopped cell proliferation, the viability was > 95%. After 3 h, two autophagy markers (SQSTM-1 expression and LC3ß localization) were altered, and at 48 h increased expression of SQSTM-1 (60%) and beclin-1 (50-100%) were found. At 24 h, the expression of endoplasmic reticulum (ER) stress markers ATF-6 and IRE-1 were increased in 100% and 150%, respectively, accompanied by XBP-1 splicing and nuclear translocation. At 48 h, major ultrastructure abnormalities were found, including ER enlargement and cytoplasmic vacuolation which was not prevented by protein synthesis inhibition. Increased PHB (85% and 40% for Tl(I) and Tl(III), respectively) and decreased ß-tubulin (45%) expression were found which may be related to the promotion of paraptosis. In summary, Tl(I) and Tl(III) promoted ER stress and probably paraptosis in MDCK cells, impairing their proliferation.

Thallium Toxicity in Caenorhabditis elegans: Involvement of the SKN-1 Pathway and Protection by S-Allylcysteine.

Monovalent thallium (Tl+) is a cation that can exert complex neurotoxic patterns in the brain by mechanisms that have yet to be completely characterized. To learn more about Tl+ toxicity, it is necessary to investigate its major effects in vivo and its ability to trigger specific signaling pathways (such as the antioxidant SKN-1 pathway) in different biological models. Caenorhabditis elegans (C. elegans) is a nematode constituting a simple in vivo biological model with a well-characterized nervous system, and high genetic homology to mammalian systems. In this study, both wild-type (N2) and skn-1 knockout (KO) mutant C. elegans strains subjected to acute and chronic exposures to Tl+ [2.5-35 µM] were evaluated for physiological stress (survival, longevity, and worm size), motor alterations (body bends), and biochemical changes (glutathione S-transferase regulation in a gst-4 fluorescence strain). While survival was affected by Tl+ in N2 and skn-1 KO (worms lacking the orthologue of mammalian Nrf2) strains in a similar manner, the longevity was more prominently decreased in the skn-1 KO strain compared with the wild-type strain. Moreover, chronic exposure led to a greater compromise in the longevity in both strains compared with acute exposure. Tl+ also induced motor alterations in both skn-1 KO and wild-type strains, as well as changes in worm size in wild-type worms. In addition, preconditioning nematodes with the well-known antioxidant S-allylcysteine (SAC) reversed the Tl+-induced decrease in survival in the N2 strain. GST fluorescent expression was also decreased by the metal in the nematode, and recovered by SAC. Our results describe and validate, for the first time, features of the toxic pattern induced by Tl+ in an in vivo biological model established with C. elegans, supporting an altered redox component in Tl+ toxicity, as previously described in mammal models. We demonstrate that the presence of the orthologous SKN-1 pathway is required for worms in evoking an efficient antioxidant defense. Therefore, the nematode represents an optimal model to reproduce mammalian Tl+ toxicity, where toxic mechanisms and novel therapeutic approaches of clinical value may be successfully pursued.

Concentration-dependent effects of sodium cholate and deoxycholate bile salts on breast cancer cells proliferation and survival.

Bile acids (BAs) are bioactive molecules that have potential therapeutic interest and their derived salts are used in several pharmaceutical systems. BAs have been associated with tumorigenesis of several tissues including the mammary tissue. Therefore, it is crucial to characterize their effects on cancer cells. The objective of this work was to analyse the molecular and cellular effects of the bile salts sodium cholate and sodium deoxycholate on epithelial breast cancer cell lines. Bile salts (BSs) effects over breast cancer cells viability and proliferation were assessed by MTS and BrdU assays, respectively. Activation of cell signaling mediators was determined by immunobloting. Microscopy was used to analyze cell migration, and cellular and nuclear morphology. Interference of membrane fluidity was studied by generalized polarization and fluorescence anisotropy. BSs preparations were characterized by transmission electron microscopy and dynamic light scattering. Sodium cholate and sodium deoxycholate had dual effects on cell viability, increasing it at the lower concentrations assessed and decreasing it at the highest ones. The increase of cell viability was associated with the promotion of AKT phosphorylation and cyclin D1 expression. High concentrations of bile salts induced apoptosis as well as sustained activation of p38 and AKT. In addition, they affected cell membrane fluidity but not significant effects on cell migration were observed. In conclusion, bile salts have concentration-dependent effects on breast cancer cells, promoting cell proliferation at physiological levels and being cytotoxic at supraphysiological ones. Their effects were associated with the activation of kinases involved in cell signalling.

Extracellular vesicles containing the transferrin receptor as nanocarriers of apotransferrin.

Previous work by our group has shown the pro-differentiating effects of apotransferrin (aTf) on oligodendroglial cells in vivo and in vitro. Further studies showed the remyelinating effect of aTf in animal demyelination models such as hypoxia/ischemia, where the intranasal administration of human aTf provided brain neuroprotection and reduced white matter damage, neuronal loss, and astrogliosis in different brain regions. These data led us to search for a less invasive and controlled technique to deliver aTf to the CNS. To such end, we isolated extracellular vesicles (EVs) from human and mouse plasma and different neuron and glia conditioned media and characterized them based on their quality, quantity, identity, and structural integrity by western blot, dynamic light scattering, and scanning electron microscopy. All sources yielded highly pure vesicles whose size and structures were in keeping with previous literary evidence. Given that, remarkably, EVs from all sources analyzed contained Tf receptor 1 (TfR1) in their composition, we employed two passive cargo-loading strategies which rendered successful EV loading with aTf, specifically through binding to TfR1. These results unveil EVs as potential nanovehicles of aTf to be delivered into the CNS parenchyma, and pave the way for further studies into their possible clinical application in the treatment of demyelinating diseases.

Gene identification and functional characterization of a Δ12 fatty acid desaturase in Tetrahymena thermophila and its influence in homeoviscous adaptation to low temperature.

Homeoviscous adaptation in poikilotherms is based in the regulation of the level of desaturation of fatty acids, variation in phospholipids head groups and sterol content in the membrane lipids, in order to maintain the membrane fluidity in response to changes in environmental temperature. Increased proportion of unsaturated fatty acids is thought to be the main response to low-temperature acclimation, which is mostly achieved by fatty acid desaturases. Genome analysis of the ciliate Tetrahymena thermophila and a gene knockout approach has allowed us to identify one Δ12 FAD and to study its activity in the original host and in a yeast heterologous expression system. The "PUFA index" -relative content of polyunsaturated fatty acids compared to the sum of saturated and monounsaturated fatty acid content- was ~57% lower at 15 °C and 35 °C in the Δ12 FAD gene knockout strain (KOΔ12) compared to WT strain. We characterized the role of T. thermophila Δ12 FAD on homeoviscous adaptation and analyzed its involvement in cellular growth, cold stress response, and membrane fluidity, as well as its expression pattern during temperature shifts. Although these alterations allowed normal growth in the KOΔ12 strain at 30 °C or higher temperatures, growth was impaired at temperatures of 20 °C or lower, where homeoviscous adaptation is impaired. These results stress the importance of Δ12 FAD in the regulation of cold adaptation processes, as well as the suitability of T. thermophila as a valuable model to investigate the regulation of membrane lipids and evolutionary conservation and divergence of the underlying mechanisms.

Effects of polyamines on cadmium- and copper-mediated alterations in wheat (Triticum aestivum L) and sunflower (Helianthus annuus L) seedling membrane fluidity.

We investigated if wheat (Wh) and sunflower (Sf) plants watering with 1 mM CdCl2 or CuCl2 for 5-15 d during germination and seedling altered membrane fluidity (MF) of their leaves and roots, and if plant pre-treatment with the polyamines (PAs) putrescine (Put), spermidine (Spd) or spermine (Spm) prevented those alterations. Cd impaired Wh and Sf growth, while Cu only affected Sf growth. Cu and Cd increased MF of leaves of both plant species, while Cd decreased MF of Sf roots. Plant treatment for 15 d with 0.1 mM Put, Spd or Spm did not affect plant growth and had opposed effects on the MF of both plants. Finally, Wh and Sf were pre-treated with PAs for either 5 or 10 days followed by metal treatment until day 15. While Put did not affect membrane MF, Spd and Spm decreased it between 5 and 10 d of plant treatment. Together, experimental results demonstrate that during plant development (a) Cd and Cu have noxious effects on plants membrane biophysical properties that could be partially responsible of their toxicity, and (b) this deleterious effect could be only partially prevented by plant pretreatment with the PAs.

Selectivity of plasma membrane calcium ATPase (PMCA)-mediated extrusion of toxic divalent cations in vitro and in cultured cells.

In the recent years, the toxicity of certain divalent cations has been associated with the alteration of intracellular Ca2+ homeostasis. Among other mechanisms, these cations may affect the functionality of certain Ca2+-binding proteins and/or Ca2+ pumps. The plasma membrane calcium pump (PMCA) maintains Ca2+ homeostasis in eukaryotic cells by mediating the efflux of this cation in a process coupled to ATP hydrolysis. The aim of this work was to investigate both in vitro and in cultured cells if other divalent cations (Sr2+, Ba2+, Co2+, Cd2+, Pb2+ or Be2+) could be transported by PMCA. Current results indicate that both purified and intact cell PMCA transported Sr2+ with kinetic parameters close to those of Ca2+ transport. The transport of Pb2+ and Co2+ by purified PMCA was, respectively, 50 and 75% lower than that of Ca2+, but only Co2+ was extruded by intact cells and to a very low extent. In contrast, purified PMCA-but not intact cell PMCA-transported Ba2+ at low rates and only when activated by limited proteolysis or by phosphatidylserine addition. Finally, purified PMCA did not transport Cd2+ or Be2+, although minor Be2+ transport was measured in intact cells. Moreover, Cd2+ impaired the transport of Ca2+ through various mechanisms, suggesting that PMCA may be a potential target of Cd2+-mediated toxicity. The differential capacity of PMCA to transport these divalent cations may have a key role in their detoxification, limiting their noxious effects on cell homeostasis.

Early response of glutathione- and thioredoxin-dependent antioxidant defense systems to Tl(I)- and Tl(III)-mediated oxidative stress in adherent pheochromocytoma (PC12adh) cells.

Thallium (Tl) is a toxic heavy metal that causes oxidative stress both in vitro and in vivo. In this work, we evaluated the production of oxygen (ROS)- and nitrogen (RNS)-reactive species in adherent PC12 (PC12adh) cells exposed for 0.5-6 h to Tl(I) or Tl(III) (10-100 µM). In this system, Tl(I) induced mostly H2O2 generation while Tl(III) induced H2O2 and ONOO·- generation. Both cations enhanced iNOS expression and activity, and decreased CuZnSOD expression but without affecting its activity. Tl(I) increased MnSOD expression and activity but Tl(III) decreased them. NADPH oxidase (NOX) activity remained unaffected throughout the period assessed. Oxidant levels returned to baseline values after 6 h of incubation, suggesting a response of the antioxidant defense system to the oxidative insult imposed by the cations. Tl also affected the glutathione-dependent system: while Tl(III) increased glutathione peroxidase (GPx) expression and activity, Tl(I) and Tl(III) decreased glutathione reductase (GR) expression. However, GR activity was mildly enhanced by Tl(III). Finally, thioredoxin-dependent system was evaluated. Only Tl(I) increased 2-Cys peroxiredoxins (2-Cys Prx) expression, although both cations increased their activity. Tl(I) increased cytosolic thioredoxin reductase (TrxR1) and decreased mitochondrial (TrxR2) expression. Tl(III) had a biphasic effect on TrxR1 expression and slightly increased TrxR2 expression. Despite of this, both cations increased total TrxR activity. Obtained results suggest that in Tl(I)-exposed PC12adh cells, there is an early response to oxidative stress mainly by GSH-dependent system while in Tl(III)-treated cells both GSH- and Trx-dependent systems are involved.

Anthocyanins inhibit tumor necrosis alpha-induced loss of Caco-2 cell barrier integrity.

An increased permeability of the intestinal barrier is proposed as a major event in the pathophysiology of conditions characterized by chronic gut inflammation. This study investigated the capacity of pure anthocyanins (AC), and berry and rice extracts containing different types and amounts of AC, to inhibit tumor necrosis alpha (TNFα)-induced permeabilization of Caco-2 cell monolayers. Caco-2 cells differentiated into intestinal epithelial cell monolayers were incubated in the absence/presence of TNFα, with or without the addition of AC or AC-rich plant extracts (ACRE). AC and ACRE inhibited TNFα-induced loss of monolayer permeability as assessed by changes in transepithelial electrical resistance (TEER) and paracellular transport of FITC-dextran. In the range of concentrations tested (0.25-1 µM), O-glucosides of cyanidin, and delphinidin, but not those of malvidin, peonidin and petunidin protected the monolayer from TNFα-induced decrease of TEER and increase of FITC-dextran permeability. Cyanidin and delphinidin acted by mitigating TNFα-triggered activation of transcription factor NF-κB, and downstream phosphorylation of myosin light chain (MLC). The protective actions of the ACRE on TNFα-induced TEER increase was positively correlated with the sum of cyanidins and delphinidins (r2 = 0.83) content in the ACRE. However, no correlation was observed between TEER and ACRE total AC, malvidin, or peonidin content. Results support a particular capacity of cyanidins and delphinidins in the protection of the intestinal barrier against inflammation-induced permeabilization, in part through the inhibition of the NF-κB pathway.

Interactions of flavan-3-ols and procyanidins with membranes: mechanisms and the physiological relevance.

Flavonoids are a type of phenolic compound widely present in edible plants. A great number of health benefits have been ascribed to flavonoid consumption in the human population. However, the molecular mechanisms involved in such effects remain to be identified. The flavan-3-ols (-)-epicatechin and (+)-catechin, and their related oligomers (procyanidins) have been thoroughly studied because of their capacity to interact with cell membranes. Starting with these interactions, procyanidins could modulate multiple biochemical processes, such as enzyme activities, receptor-ligand binding, membrane-initiated cell signaling, and molecule transport across membranes. This review focuses on molecular aspects of procyanidin interactions with membrane lipid components, and the resulting protection of the membranes against mechanical and/or oxidative damage, resulting in the maintenance of cell functions.

Tl(I) and Tl(III) alter the expression of EGF-dependent signals and cyclins required for pheochromocytoma (PC12) cell-cycle resumption and progression.

The effects of thallium [Tl(I) and Tl(III)] on the PC12 cell cycle were evaluated without (EGF(-)) or with (EGF(+)) media supplementation with epidermal growth factor (EGF). The following markers of cell-cycle phases were analyzed: cyclin D1 (G1 ); E2F-1, cyclin E and cytosolic p21 (G1 →S transition); nuclear PCNA and cyclin A (S); and cyclin B1 (G2). The amount of cells in each phase and the activation of the signaling cascade triggered by EGF were also analyzed. Tl(I) and Tl(III) (5-100 µM) caused dissimilar effects on PC12 cell proliferation. In EGF(-) cells, Tl(I) increased the expression of G1 →S transition markers and nuclear PCNA, without affecting cyclin A or cyclin B1. In addition to those, cyclin B1 was also increased in EGF(+) cells. In EGF(-) cells, Tl(III) increased the expression of cyclin D1, all the G1→S and S phase markers and cyclin B1. In EGF(+) cells, Tl(III) increased cyclin D1 expression and decreased all the markers of G1 →S transition and the S phase. Even when these cations did not induce the activation of EGF receptor (EGFR) in EGF(-) cells, they promoted the phosphorylation of ERK1/2 and Akt. In the presence of EGF, the cations anticipated EGFR phosphorylation without affecting the kinetics of EGF-dependent ERK1/2 and Akt phosphorylation. Altogether, results indicate that Tl(I) promoted cell proliferation in both EGF(-) and EGF(+) cells. In contrast, Tl(III) promoted the proliferation of EGF(-) cells but delayed it in EGF(+) cells, which may be related to the toxic effects of this cation in PC12 cells.

Procyanidins can interact with Caco-2 cell membrane lipid rafts: involvement of cholesterol.

Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-ß-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol-Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex-cholesterol bondings. Procyanidin-lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes.

BACKGROUND: The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). METHODS: Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. RESULTS: Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10µM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. CONCLUSIONS: MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. GENERAL SIGNIFICANCE: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

Prunin- and hesperetin glucoside-alkyl (C4-C18) esters interaction with Jurkat cells plasma membrane: consequences on membrane physical properties and antioxidant capacity.

Prunin (P)- and hesperetin glucoside (HG)-alkyl esters are lipid-soluble compounds with antimicrobial and antioxidant capacities in vitro. The effects of P- and HG-alkyl (C4-C18) esters (0.1-100µM) on human leukemia T (Jurkat) cells viability and plasma membrane fluidity were evaluated. After 1h of exposure, cell viability was not affected in the range 0.1-10µM. The decrease of cell viability found at 100µM concentration depended on the length of the alkyl chain and reached a maximum with C6-C12 derivatives. At this concentration, cell hyperpolarization and shrinkage were also observed. Cell plasma membrane fluidity was not affected, regardless the depths of the membrane level evaluated, but mild changes in plasma membrane hydration were found. Esterification did not affect the antioxidant capacity of P and HG (0.1-10µM) against 1mM H2O2. When exposed to 1mM AAPH, P-alkyl esters retained P antioxidant capacity, but HG-derivatives acted as pro-oxidants. Together, present experimental evidences suggest that short term exposures to 0.1-10µM concentrations of P- and HG-alkyl (C4-C18) esters can be considered safe for cultured human cells, and further studies are required to investigate their long term effects, as well their safety for human consumption.

Endosomes and lysosomes are involved in early steps of Tl(III)-mediated apoptosis in rat pheochromocytoma (PC12) cells.

The mechanisms that mediate thallium (Tl) toxicity are still not completely understood. The exposure of rat pheochromocytoma (PC12) cells to Tl(I) or Tl(III) activates both mitochondrial (Tl(I) and Tl(III)) and extrinsic (Tl(III)) pathways of apoptosis. In this work we evaluated the hypothesis that the effects of Tl(III) may be mediated by the damage to lysosomes, where it might be incorporated following the route of iron uptake. PC12 cells exposed for 3 h to 100 µM Tl(III) presented marked endosomal acidification, effect that was absent when cells were incubated in a serum-free medium and that was fully recovered when the latter was supplemented with transferrin. After 6 h of incubation the colocalization of cathepsins D and B with the lysosomal marker Lamp-1 was decreased together with an increase in the total activity of the enzymes. A permanent damage to lysosomes after 18 h of exposure was evidenced from the impairment of acridine orange uptake. Cathepsin D caused the cleavage of pro-apoptotic protein BID that is involved in the activation of the intrinsic pathway of apoptosis. Supporting that, BID cleavage and the activation of caspase 3 by Tl(III) were fully prevented when cells were preincubated with cathepsin D inhibitor (pepstatin A) and only partially prevented when cathepsin B inhibitor (E64d) was used. None of these inhibitors affected BID cleavage or caspase 3 activation in Tl(I)-treated cells. Together, experimental results support the role of Tl(III) uptake by the acidic cell compartments and their involvement in the early steps of Tl(III)-mediated PC12 cells apoptosis.

Large procyanidins prevent bile-acid-induced oxidant production and membrane-initiated ERK1/2, p38, and Akt activation in Caco-2 cells.

Procyanidins are oligomers of flavanol subunits present in large amounts in fruits and vegetables. Their consumption is associated with health benefits against colonic inflammation and colorectal cancer (CRC). Large procyanidins (with more than three subunits) are not absorbed by intestinal epithelial cells but could exert biological actions through their interactions with the cell membrane. This study investigated the capacity of hexameric procyanidins (Hex) to prevent oncogenic events initiated by deoxycholic acid (DCA), a secondary bile acid linked to the promotion of CRC. Hex interacted with Caco-2 cell membranes preferentially at the water-lipid interface. Hex (2.5-20 µM) inhibited DCA-triggered increase in cellular calcium, NADPH oxidase activation, and oxidant production. DCA promoted the activation of protein kinase B (Akt), of the mitogen-activated protein kinases ERK1/2 and p38, and of the downstream transcription factor AP-1. This activation was not triggered by calcium or oxidant increases. Hex caused a dose-dependent inhibition of DCA-mediated activation of all these signals. DCA also triggered alterations in the cell monolayer morphology and apoptotic cell death, events that were delayed by Hex. The capacity of large procyanidins to interact with the cell membrane and prevent those cell membrane-associated events can in part explain the beneficial effects of procyanidins on CRC.

Basic biochemical mechanisms behind the health benefits of polyphenols.

Polyphenols and consequently many flavonoids have several beneficial actions on human health. However, the actual molecular interactions of polyphenols with biological systems remain mostly speculative. This review addresses the potential mechanisms of action that have been so far identified, as well as the feasibility that they could occur in vivo. Those mechanisms include: i) non specific actions, based on chemical features common to most polyphenols, e.g. the presence of a phenol group to scavenge free radicals; and ii) specific mechanisms; based on particular structural and conformational characteristics of select polyphenols and the biological target, e.g. proteins, or defined membrane domains. A better knowledge about the nature and biological consequences of polyphenol interactions with cell components will certainly contribute to develop nutritional and pharmacological strategies oriented to prevent the onset and/or the consequences of human disease.

The plasma membrane plays a central role in cells response to mechanical stress.

The mechanisms by which lymphocytes recognize and interpret mechanical stimuli and translate these into the triggering of select signaling cascades that are critical for lymphocyte function are still not fully understood. In this work, we investigated the association of mechanical stress (MS)-induced changes in membrane physical properties with changes in cytoskeleton dynamics and cell signaling. In Jurkat T cells, MS was associated with the immediate and transient depolymerization of both beta-tubulin and F-actin. The fluidity of the plasma membrane measured in the hydrophobic region of the bilayer, increased 0.5 min post-MS, recovering the initial value in the following 2 min. This effect was accompanied by the rearrangement of lipids in the lateral phase of the plasma membrane, transient lipid rafts' alteration, and membrane hyperpolarization. The consequent increase in cellular [Ca2+] triggered the activation of the transcription factors NFAT, AP-1, and NF-kappaB. Results indicate that the cytoplasmic membrane, through changes in membrane physical properties, senses MS, and transduces an initial physical stimulus into microtubules rearrangements, Ca2+ mobilization, and the subsequent changes in cell signaling.

Antioxidant actions of flavonoids: thermodynamic and kinetic analysis.

The benefits of flavonoids on human health are very often ascribed to their potential ability to act diminishing free radical steady state concentration in biological systems providing antioxidant protection. This is an assumption based on the chemical structures of flavonoids that support their capacity to scavenge free radicals and chelate redox-active metals. In this paper we will use thermodynamic and kinetic approaches to analyze the interactions of flavonoids with biological material and from there, extrapolate the physiological relevance of their antioxidant actions. Thermodynamic analysis predicts that both, scavenging of oxygen-derived radicals and the sequestration of redox-active metals are energetically favored. Nevertheless, the actual concentrations reached by flavonoids in most animal and human tissues following dietary ingestion are incompatible with the kinetic requirements necessary to reach reaction rates of physiological relevance. This incompatibility becomes evident when compared to other antioxidant compounds, e.g. alpha-tocopherol (vitamin E), ascorbate (vitamin C), and glutathione. Alternatively, lipid-flavonoid and protein-flavonoid interactions can indirectly mediate a decrease in oxidant (free radical) production and/or oxidative damage to both cell and extracellular components. The final mechanisms mediating the antioxidant actions of flavonoid will be determined by their actual concentration in the tissue under consideration.