1,721,036 research outputs found
Intracellular antioxidant activities of polyphenolic compounds (single and in mixture): resveratrol and quercitin
No full textUnder physiopathological conditions reactive oxygen species can be over produced evoking an intracellular state of oxidative stress. Dietary polyphenols are known to have potent antioxidant activity by various mechanisms. While a number of data is available on the antioxidant behavior of single polyphenolic compounds, little is known about possible interactions among them, which could influence the “quality” of their intracellular antioxidant activity when present in mixture.
This study was aimed to assess the intracellular antioxidant activity of two red wine polyphenols, resveratrol and quercetin, both single and in mixture. HeLa cells charged with the ROS-sensitive cell permeant dye, CM-H2DCFDA, were used as model. Spectrofluorimetric and confocal microscopy analysis was performed.
In the range of concentration tested (5-50 μM) quercetin alone exhibited a dose-dependent intracellular antioxidant activity against an oxidative stress induced by H2O2 used as pro-oxidant agent. On the other hand resveratrol showed a pro-oxidant intracellular activity. When the cells were exposed to equimolar mixtures of the two compounds, an intracellular interaction among them was highlighted, with a non-predictable behavior on the basis of the analysis conducted on the single compounds.
In conclusion results demonstrated that antioxidant properties of polyphenolic compounds depend not only on the nature of the compound itself, but also on the type of their interaction in a cellular system
Cloride transport in HeLa cells: a study by a recombinant cell-based assay in living cells
No full textChloride transport is involved in a wide variety of cellular functions, including regulation of the membrane potential, cell volume, acid base balance, and epithelial fluid absorption and secretion. Alterations in chloride transport mechanisms are related to a number of physio-pathological conditions.
The aim of this study was to investigate chloride transport across the plasma membrane of cervical cancer cell lines (HeLa), relying on the halide binding properties of a green (GFP) fluorescent protein variant (YFP) that, if halide complexed, decreases the basal fluorescence intensity. The fluorescence changes of HeLa cells expressing YFP was analyzed by confocal and spectrofluorometric analysis.
Our results highlights the presence of a transmembrane halide transport through fluorescence quenching when the transfected cells were exposed to NaI, used as a surrogate ion, under isotonic conditions. This quenching was inhibited by bumetanide, a specific inhibitor of Na+-K+-2Cl- cotransporter, demonstrating the role of such transporter in intracellular halide absorption and allowing cellular fluorescence assay for a functional study of Na+-K+-2Cl-. The kinetic parameters of the transporter were quantified.
The present work provides an alternative approach for the measurement and characterization of Cl- and halide transport in living cells expressing YFP-H148Q/I152L based on a non-invasive analysis method
Transepithelial absorption and intracellular antioxidant activity of hydroxytyrosol in rat intestine
No full textHydroxytyrosol is the olive natural polyphenol with the highest free radical scavenger activity ever reported for any natural compound. The aim of this work was: 1) to investigate the mechanisms involved in hydroxytyrosol intestinal transport, using Ussing chamber mounted rat jejunum; 2) to investigate hydroxytyrosol intracellular antioxidant activity in intestinal cells, by confocal microscopy on native tissue charged with the fluorescent probe CM-H2- DCFDA.
A mucosa-serosa flux of hydroxytyrosol was demonstrated in rat jejunum. It showed a saturating kinetic with a Km apparent of 4.34 μM and a Vmax of 13.47 nmol/min/cm2. No significant effect was observed on transepithelial potential, short circuit current, and transepithelial resistance. The intracellular antioxidant activity was determined on rat colon segments incubated with different hydroxytyrosol concentrations, charged with the fluorescent probe, then exposed to H2O2 as oxidant agent and observed by confocal microscopy. Hydroxytyrosol was able to significantly inhibit ROS production in surface colonocytes either in basal or under oxidative stress condition in a dose-dependent manner.
In conclusion, a specific transepithelial hydroxytyrosol transport was demonstrated in rat jejunum. In intestinal cells hydroxytyrosol was demonstrated to exert a significant antioxidant intracellular activity suggesting a possible protecting effect on intestinal mucosa from pathological conditions related to oxidative stress
Hypertonicity stimulates Cl- transport in the intestine of fresh water acclimated eel, Anguilla anguilla
No full textEel intestinal epithelium when bathed symmetrically with normal Ringer solution develops a net Cl- current (short circuit current, Isc) giving rise to a negative transepithelial potential (Vt) at the basolateral side of the epithelium, lower in fresh-water (FW)-acclimated animals with respect to sea-water (SW). The aim of the present work was to study the cell response to hypertonic stress of FW eel intestinal epithelium in relation to Cl- absorption. The hypertonicity of the external bathing solutions produced first a transient increase of Vt and Isc, then followed (after10-15min) by a gradual and sustained increase which reached the maximum value after 40-60 min. The morphometric analysis of the intestine revealed the shrinkage of the cells after 5 min hypertonicity exposure, and then a regulatory volume increase (RVI) response, which parallels the gradual and sustained increase in the electrophysiological parameters. This last phase is inhibited by drugs known to block Cl- absorption in eel intestine, such as luminal bumetanide (10 M), specific inhibitor of Na+-K+-2Cl- cotransport, or basolateral NPPB (0.5 mM), dichloro-DPC (0.5 mM), inhibitors of basolateral Cl- conductance. Serosal dimethyl-amiloride (100 M), specific inhibitor of the Na+/H+ antiport, was ineffective on the hyperosmotic response.
Bicarbonate revealed a crucial role as a modulator of hypertonicity response, since in bicarbonate-free conditions or in the presence of serosal 0.25 mM SITS, blocker of HCO3- transport systems, the Isc response to hypertonicity was lost. In nominally Ca2+-free conditions the Isc response to hypertonicity was abolished. The same results were obtained by bilateral addition of 100 M verapamil or 50 M nifedipine or 1 mM lanthanum, known Ca2+ channel blockers, indicating that extracellular Ca2+ plays a key role for the activation of Cl- current in the response to hypertonic stress.
The data show that in the eel intestinal epithelium the hypertonicity of the external medium affects cell volume which in turn might represent the signal to increase the rate of Cl- transport. This response is sustained by the activation of the luminal Na+-K+-2Cl- cotransporter and the functionality of basolateral Cl- channels
Cell volume regulation and apoptotic volume decrease in isolated eel enterocytes.
No full textAim: Programmed cell death is a physiological form of death that is crucial in growth, maintenance, and normal development of tissues. Despite the universal prominence of cell volume loss in cells undergoing apoptosis, the relationship between this event and ensuing cell death remains uncertain. The aim of the present work was to study the role of cell volume regulation in the Apoptotic Volume Decrease (AVD), which represents one of the first events in apoptosis. The study was carried out on enterocytes isolated from the intestine of the teleost European eel. In the intact tissue the ion transport mechanisms responsible for cell volume regulation have been previously characterized (Lionetto et al., Cell Physiol. Biochem., 11: 41-54, 200; Lionetto et al., J. Exp. Biol., 208: 749-760, 2005; Lionetto et al., Cell Physiol. Biochem., 21: 361-372, 2008).
Methods: Video imaging was utilized for cell volume measurement, while confocal microscopy was applied on cells labelled with annexin V for monitoring the onset of apoptosis.
Results: Isolated enterocytes performed Regulatory Volume Decrease (RVD) following hypotonic swelling by the activation of high conductance Ca2+ activated K+ channels (BK channels). On the other hand, they were unable to perform Regulatory Volume Increase (RVI) following hypertonic shrinkage. The cells underwent AVD (corresponding to a cell shrinkage in isotonic condition) after 4-5 hours after isolation. The phenomenon was reversed by iberiotoxin, specific inhibitor of BK channels. Iberiotoxin was also able to delay the onset of apoptosis.
Conclusions: Induction of the apoptotic volume decrease (AVD) under normotonic conditions shares cellular mechanisms that are used by the cells in response to osmotic changes. In isolated eel enterocytes AVD is dependent on BK activated K+ efflux and it is facilitated by the inactivation of ion transport mechanisms involved in RVI
Induction of the apoptotic volume decrease (AVD) under normotonic conditions in HeLa cells exposed to Trolox
No full textPrevious observations showed that Trolox, a synthetic analog of vitamin E, widely used as antioxidant standard in a number of bioassays, can exert a pro-oxidant behavior at higher concentrations (>40 μM) on HeLa cells exposed for 24h, producing an isotonic cell shrinkage. A number of cellular events are known to be triggered by oxidative stress, including impairment of ion transport mechanisms and alteration of cell volume homeostasis.
This work aims to investigate the possible mechanisms through which Trolox at high concentrations acts on cell volume homeostasis alteration in HeLa cells.
The study was carried out by 1) spectrofluorimetric determination of intracellular oxidative stress in cells charged with CM-H2DCFDA, 2) morphometric analysis of cells observed under optical microscopy for cell volume determination, and 3) spectrofluorimetric and confocal analysis of cells charged with Annexin V/Propidium Iodide for apoptotic induction.
HeLa cells exposed for 24h to high Trolox concentrations showed a significant dose-dependent isotonic reduction of cell volume associated to intracellular oxidative stress. The observed isotonic shrinkage was accompanied by apoptosis induction, as demonstrated by Annexin V/Propidium Iodide and was ascribed to Apoptotic Volume Decrease (AVD).
The isotonic shrinkage appearance was demonstrated to occur early (after 2h) during the exposure to high Trolox concentrations. It was completely inhibited by pretreatment of the cells with a Cl- channel blocker SITS (0.5 mM).
These results indicate that treatment of HeLa cells with high Trolox concentrations induces the activation of volume-regulatory Cl- channels, most likely by an increase in endogenous ROS production, which in turn is able to generate AVD
Intracellular antioxidant activity of hydroxytyrosol in rat colon epithelium
No full textHydroxytyrosol (HT) is a phenolic compound occurring in olive leaves and pulp, known for its antioxidant activity. It is significantly present in olive oil, which is the most diffused fat source in Mediterranean countries. The work is aimed to improve the knowledge about the direct biological effects of HT on the intestinal mucosa, which is the district of the body exposed to the highest HT concentrations through the diet intake. In particular, the work is addressed to explore the antioxidant activity of intracellularly absorbed HT on colon epithelium, which is one of the gastrointestinal tract segments more exposed to pro-oxidant conditions.
The study was carried out by in situ confocal microscopy on rat colon explants, loaded with the ROS sensitive fluorescent probe CM-H2DCFDA, under basal physiological and pro-oxidant conditions.
HT was able to dose-dependently decrease the intracellular reactive oxygen species (ROS) production under basal physiological conditions in superficial colonocytes. Moreover, it produced a direct dose-dependent antioxidant protective effect on the colon epithelium exposed to a pro-oxidant condition such as H2O2 exposure. Moreover, the antioxidant effect of HT was significantly expressed also against the oxidative stress induced by another pro-oxidant agent, ter-butyl-peroxyde.
The results demonstrated that HT can positively contribute to the redox balance of colon epithelium by decreasing intracellular ROS either in basal or pro-oxidant conditions, suggesting the potential use of HT as a functional food ingredient useful for the protection of the intestinal mucosa against oxidative stress
A novel physiological role for carbonic anhydrase in animals: involvement in the lysosomal response to cadmium exposure in mussel digestive gland
No full textCarbonic anhydrase (CA) is a ubiquitous metalloenzyme involved in a number of physiological processes including respiration, pH homeostasis, electrolyte transport, calcification, biosynthetic reactions.
The aim of this work was to explore a novel functional role of CA in animals: its involvement in the lysosomal system response to metal exposure. Mytilus galloprovincialis digestive gland was used as experimental model.
The in vivo exposure of mussels to CdCl2 was able to evoke the induction of digestive gland CA. This response was paralleled by the activation of the lysosomal compartment (proliferation and/or increase in size of lysosomes), as assessed on LysoSensor Green marked cells by confocal microscopy and spectrofluorimetric analysis. When mussels were in vivo exposed to acetazolamide, specific CA inhibitor, the acidification of the lysosomal compartment was prevented. Moreover, during the co-exposure to cadmium and acetazolamide, the cadmium induced activation of the lysosomal compartment was completely inhibited.
In conclusion, our results demonstrated in vivo the physiological contribution of CA to the acidification of the lysosomal compartment in digestive gland and its involvement in the lysosomal activation following cadmium exposure. CA induction following cadmium exposure could physiologically sustain a prolonged increased requirement of H+ for supporting lysosomal acidification during lysosomal activation
Carbonic anhydrase activity in the digestive gland of Mytilus galloprovincialis: the story of a novel biomarker?
No full textAim: Carbonic anhydrase is a zinc metalloenzyme catalysing the reversible hydration of CO2. Its activity is virtually ubiquitous in nature and is involved in a wide variety of physiological processes. The aim of the present work was to study the in vitro and in vivo sensitivity of CA to chemical pollutants in Mytilus galloprovincialis, widely used as sentinel organism in marine pollution biomonitoring, with the aim to develop a new biomarker of exposure/effect to chemical contamination.
Methods: CA activity was measured applying a simple and low cost electrometric method. Briefly, enzymatic activity units were calculated from the rate of H+ production in the reaction mixture (where CO2 as substrate was present) against a blank containing the specific CA inhibitor acetazolamide.
Results: As first step, we demonstrated the presence of a significant CA activity in M. galloprovincialis tissues and its sensitivity to in vitro exposure to several heavy metals. As second step, we demonstrated a significant tissue-specific alteration of CA activity following in vivo Cadmium exposure, with a significant increase in the digestive gland. As third step, immunoprecipitation and western blot analysis on mussel digestive gland revealed a protein that immunoreacts with the antibody at a molecular mass of approximate 42 kDa (the sequencing analysis is in progress). Interestingly the 42 KDa band, ascribed to CA, appeared strongly increased in the digestive gland of cadmium exposed mussel (14 day exposure), thus leading to an induction of CA expression in accordance with the observed enzymatic activity increase. As final step, the laboratory resulted was validated with field experiments.
Conclusion: Obtained results represent a good starting point for future potential biomarker application of the CA activity in the sentinel organism M. galloprovincialis
Biomarker application for the study of chemical contamination risk on marine organisms in the Taranto marine coastal area
No full textThis work represents a pilot study for monitoring the potential toxicological risk of commercial relevant marine resources along the South coast of Italy by using biomarkers as complementary tool to chemical analysis. The attention was focused on the industrialized area of Salento peninsula, such as Taranto, that, in spite of the presence of the big industry (oil, metal industry), sustains activities related to the sea resources, such as fishery and mussel-culture. The study was carried out in fish, such as Mullus barbatus and Trachurus mediterraneus, two important fish species for the fishery in this area, and in mussels (Mytilus galloprovincialis). As control area S. Maria di Leuca, area of naturalistic interest, was chosen.
In fish, liver metallothionein levels (specific index of exposure to heavy metals such as Hg, Cd, Cu, Zn) and brain and muscular acetylcholinesterase activity (specific index of exposure to organophosphate and carbamate pesticides) were measured. None of the two fish species showed significant differences in acetylcholinesterase activity and in pesticide trace level between the anthropogenic impact exposed site and the control group. On the other hand, metallothionein hepatic levels in Mullus barbatus were significantly increased in the organisms coming from Taranto with respect to the organisms coming from the control site, but chemical analysis, routinely performed on edible muscle for the evaluation of chemical quality of fish products, did not reveal high heavy metal concentration in the edible muscle of fish from Taranto.
Mussels exposed for one month in the Mar Piccolo of Taranto, an important mussel farming area, showed increase in the level of catalase activity, an oxidative stress index, increase in the levels of metallothioneins and inhibition of acetylcholinesterase activity
The need to integrate chemical analysis with the study of biological responses to pollutants (biomarkers) in marine organisms is discussed for a better comprehension of the impact of chemical contaminants on the sea and its resource
- …
