Clozapine suppresses NADPH oxidase activation, counteracts cytosolic H2O2, and triggers early onset mitochondrial dysfunction during adipogenesis of human liposarcoma SW872 cells.

Long-term treatment of schizophrenia with clozapine (CLZ), an atypical antipsychotic drug, is associated with an increased incidence of metabolic disorders mediated by poorly understood mechanisms. We herein report that CLZ, while slowing down the morphological changes and lipid accumulation occurring during SW872 cell adipogenesis, also causes an early (day 3) inhibition of the expression/nuclear translocation of CAAT/enhancer-binding protein ß and peroxisome proliferator-activated receptor γ. Under the same conditions, CLZ blunts NADPH oxidase-derived reactive oxygen species (ROS) by a dual mechanism involving enzyme inhibition and ROS scavenging. These effects were accompanied by hampered activation of the nuclear factor (erythroid-derived2)-like 2 (Nrf2)-dependent antioxidant responses compared to controls, and by an aggravated formation of mitochondrial superoxide. CLZ failed to exert ROS scavenging activities in the mitochondrial compartment but appeared to actively scavenge cytosolic H2O2 derived from mitochondrial superoxide. The early formation of mitochondrial ROS promoted by CLZ was also associated with signs of mitochondrial dysfunction. Some of the above findings were recapitulated using mouse embryonic fibroblasts. We conclude that the NADPH oxidase inhibitory and cytosolic ROS scavenging activities of CLZ slow down SW872 cell adipogenesis and suppress their Nrf2 activation, an event apparently connected with increased mitochondrial ROS formation, which is associated with insulin resistance and metabolic syndrome. Thus, the cellular events characterised herein may help to shed light on the more detailed molecular mechanisms explaining some of the adverse metabolic effects of CLZ.

ERO1α primes the ryanodine receptor to respond to arsenite with concentration dependent Ca2+ release sequentially triggering two different mechanisms of ROS formation.

A 6 h exposure of U937 cells to 2.5 µM arsenite stimulates low Ca2+ release from the inositol 1, 4, 5-triphosphate receptor (IP3R), causing a cascade of causally connected events, i.e., endoplasmic reticulum oxidoreductin-1α (ERO1α) expression, activation of the ryanodine receptor (RyR), mitochondrial Ca2+ accumulation, mitochondrial superoxide formation and further ERO1α expression. At greater arsenite concentrations, the release of the cation from the IP3R and the ensuing ERO1α expression remained unchanged but were nevertheless critical to sequentially promote concentration-dependent increases in Ca2+ release from the RyR, NADPH oxidase activation and a third mechanism of ERO1α expression which, in analogy to the one driven by mitochondrial superoxide, was also mediated by reactive oxygen species (ROS) and devoid of effects on Ca2+ homeostasis. Thus, concentration-independent stimulation of Ca2+ release from the IP3R is of pivotal importance for the effects of arsenite on Ca2+ homeostasis. It stimulates the expression of a fraction of ERO1α that primes the RyR to respond to the metalloid with concentration-dependent Ca2+-release, triggering the formation of superoxide in the mitochondrial respiratory chain and via NADPH oxidase activation. The resulting dose-dependent ROS formation was associated with a progressive increase in ERO1α expression, which however failed to affect Ca2+ homeostasis, thereby suggesting that ROS, unlike IP3R-dependent Ca2+ release, promote ERO1α expression in sites distal from the RyR.

Nrf2-Mediated Pathway Activated by Prunus spinosa L. (Rosaceae) Fruit Extract: Bioinformatics Analyses and Experimental Validation.

In our previous studies, Prunus spinosa fruit (PSF) ethanol extract was showed to exert antioxidant, antimicrobial, anti-inflammatory and wound healing activities. In the present study, an integrated bioinformatics analysis combined with experimental validation was carried out to investigate the biological mechanism(s) that are responsible for the reported PSF beneficial effects as an antioxidant during a pro-inflammatory TLR4 insult. Bioinformatics analysis using miRNet 2.0 was carried out to address which biological process(es) the extract could be involved in. In addition, Chemprop was employed to identify the key targets of nuclear receptor (NR) signaling and stress response (SR) pathways potentially modulated. The miRNet analysis suggested that the PSF extract mostly activates the biological process of cellular senescence. The Chemprop analysis predicted three possible targets for nine phytochemicals found in the extract: (i) ARE signaling, (ii) mitochondrial membrane potential (MMP) and (iii) p53 SR pathways. The PSF extract antioxidant effect was also experimentally validated in vitro using the human monocyte U937 cell line. Our findings showed that Nrf2 is modulated by the extract with a consequent reduction of the oxidative stress level. This was confirmed by a strong decrease in the amount of reactive oxygen species (ROS) observed in the PSF-treated cells subjected to lipopolysaccharide (LPS) (6 h treatment, 1 µg/mL). No visible effects were observed on p53 and MMP modulation.

Arsenite enhances ERO1α expression via ryanodine receptor dependent and independent mechanisms.

Arsenite is a potent carcinogen and toxic compound inducing an array of deleterious effects via different mechanisms, which include the Ca2+-dependent formation of reactive oxygen species. The mechanism whereby the metalloid affects Ca2+ homeostasis involves an initial stimulation of the inositol 1, 4, 5-triphosphate receptor, an event associated with an endoplasmic reticulum (ER) stress leading to increased ERO1α expression, and ERO1α dependent activation of the ryanodine receptor (RyR). Ca2+ release from the RyR is then critically connected with the mitochondrial accumulation of Ca2+. We now report that the resulting formation of mitochondrial superoxide triggers a second mechanism of ER stress dependent ERO1α expression, which however fails to impact on Ca2+ release from the RyR or, more generally, on Ca2+ homeostasis. Our results therefore demonstrate that arsenite stimulates two different and sequential mechanisms leading to increased ERO1α expression with different functions, possibly due to their different subcellular compartmentalization.

Loss-of-rescue of Ryr1I4895T-related pathology by the genetic inhibition of the ER stress response mediator CHOP.

RYR1 is the gene encoding the ryanodine receptor 1, a calcium release channel of the endo/sarcoplasmic reticulum. I4898T in RYR1 is one of the most common mutations that give rise to central core disease (CCD), with a variable phenotype ranging from mild to severe myopathy to lethal early-onset core-rod myopathy. Mice with the corresponding I4895T mutation in Ryr1 present mild myopathy when the mutation is heterozygous while I4895T homozygous is perinatal-lethal. Here we show that skeletal muscles of I4895T homozygous mice at birth present signs of stress of the endoplasmic reticulum (ER stress) and of the related unfolded protein response (UPR) with increased levels of the maladaptive mediators CHOP and ERO1. To gain information on the role of CHOP in the pathogenesis of RYR1I4895T-related myopathy, we generated compound Ryr1I4895T, Chop knock-out (-/-) mice. However, the genetic deletion of Chop, although it attenuates ER stress in the skeletal muscle of the newborns, does not rescue any phenotypic or functional features of Ryr1I4895T in mice: neither the perinatal-lethal phenotype nor the inability of Ryr1I4895T to respond to its agonist caffeine, but protects from ER stress-induced apoptosis. These findings suggest that genetic deletion of the ER stress response mediator CHOP is not sufficient to counteract the pathological Ryr1I4895T phenotype.

Inhibition of activity/expression, or genetic deletion, of ERO1α blunts arsenite geno- and cyto-toxicity.

Our recent studies suggest that arsenite stimulates the crosstalk between the inositol 1, 4, 5-triphosphate receptor (IP3R) and the ryanodine receptor (RyR) via a mechanism dependent on endoplasmic reticulum (ER) oxidoreductin1α (ERO1α) up-regulation. Under these conditions, the fraction of Ca2+ released by the RyR via an ERO1α-dependent mechanism was promptly cleared by the mitochondria and critically mediated O2-. formation, responsible for the triggering of time-dependent events associated with strand scission of genomic DNA and delayed mitochondrial apoptosis. We herein report that, in differentiated C2C12 cells, this sequence of events can be intercepted by genetic deletion of ERO1α as well as by EN460, an inhibitor of ERO1α activity. Similar results were obtained for the early effects mediated by arsenite in proliferating U937 cells, in which however the long-term studies were hampered by the intrinsic toxicity of the inhibitor. It was then interesting to observe that ISRIB, an inhibitor of p-eIF2 alpha, was in both cell types devoid of intrinsic toxicity and able to suppress ERO1α expression and the resulting downstream effects leading to arsenite geno- and cyto-toxicity. We therefore conclude that pharmacological inhibition of ERO1α activity, or expression, effectively counteracts the deleterious effects induced by the metalloid via a mechanism associated with prevention of mitochondrial O2-. formation.

Mitochondrial ROS, ER Stress, and Nrf2 Crosstalk in the Regulation of Mitochondrial Apoptosis Induced by Arsenite.

Long-term ingestion of arsenicals, a heterogeneous group of toxic compounds, has been associated with a wide spectrum of human pathologies, which include various malignancies. Although their mechanism of toxicity remains largely unknown, it is generally believed that arsenicals mainly produce their effects via direct binding to protein thiols and ROS formation in different subcellular compartments. The generality of these mechanisms most probably accounts for the different effects mediated by different forms of the metalloid in a variety of cells and tissues. In order to learn more about the molecular mechanisms of cyto- and genotoxicity, there is a need to focus on specific arsenic compounds under tightly controlled conditions. This review focuses on the mechanisms regulating the mitochondrial formation of ROS after exposure to low concentrations of a specific arsenic compound, NaAsO2, and their crosstalk with the nuclear factor (erythroid-2 related) factor 2 antioxidant signaling and the endoplasmic reticulum stress response.

Crosstalk between ERO1α and ryanodine receptor in arsenite-dependent mitochondrial ROS formation.

Arsenite, a well-established human carcinogen and toxic compound, promotes the formation of mitochondrial superoxide (mitoO2-) via a Ca2+-dependent mechanism, in which an initial stimulation of the inositol 1, 4, 5-trisphosphate receptor (IP3R) is followed by the activation of the ryanodine receptor (RyR), critical for providing Ca2+ to the mitochondria. We now report that, under the same conditions, arsenite triggers endoplasmic reticulum (ER) stress and a threefold increase in ER oxidoreductin 1α (ERO1 α) levels in proliferating U937 cells. EN460, an inhibitor of ERO1 α, recapitulated all the effects associated with RyR inhibition or downregulation, including prevention of RyR-induced Ca2+ accumulation in mitochondria and the resulting O2-. formation. Quantitatively similar results were obtained in inhibitor studies performed in terminally differentiated wild type C2C12 cells. Moreover, ERO1 α knockout C2C12 myotubes responded to arsenite as their wild type counterpart supplemented with EN460. As a final note, arsenite enhanced the expression of ERO1 α via a mechanism mediated by Ca2+ release from both the IP3R and RyR. We therefore conclude that arsenite activates a positive feedback amplification cycle between Ca2+ levels and ERO1 α in the ER, by which IP3R-dependent Ca2+ induces ERO1 α and ERO1 α promotes Ca2+ release via RyR, thereby amplifying the initial Ca2+ load and causing the mitochondrial accumulation of the cation, critical for mitoO2- formation.

Functional organization of the endoplasmic reticulum dictates the susceptibility of target cells to arsenite-induced mitochondrial superoxide formation, mitochondrial dysfunction and apoptosis.

Arsenite induces many critical effects associated with the formation of reactive oxygen species (ROS) through different mechanisms. We focused on Ca2+-dependent mitochondrial superoxide (mitoO2-.) formation and addressed questions on the effects of low concentrations of arsenite on the mobilization of the cation from the endoplasmic reticulum and the resulting mitochondrial accumulation. Using various differentiated and undifferentiated cell types uniquely expressing the inositol-1, 4, 5-triphosphate receptor (IP3R), or both the IP3R and the ryanodine receptor (RyR), we determined that expression of this second Ca2+ channel is an absolute requirement for mitoO2-. formation and for the ensuing mitochondrial dysfunction and downstream apoptosis. In arsenite-treated cells, RyR was recruited after IP3R stimulation and agonist studies provided an indirect indication for a close apposition between RyR and mitochondria. It was also interesting to observe that arsenite fails to promote mitochondrial Ca2+ accumulation, mitoO2-. formation and mitochondrial toxicity in RyR-devoid cells, in which the IP3R is in close contact with the mitochondria. We therefore conclude that low dose arsenite-induced mitoO2- formation, and the resulting mitochondrial dysfunction and toxicity, are prerequisite of cell types expressing the RyR in close apposition with mitochondria.

Temporal correlation of morphological and biochemical changes with the recruitment of different mechanisms of reactive oxygen species formation during human SW872 cell adipogenic differentiation.

Human SW872 preadipocyte conversion to mature adipocytes is associated with time-dependent changes in differentiation markers' expression and with morphological changes accompanied by the accumulation of lipid droplets (LDs) as well as by increased mitochondriogenesis and mitochondrial membrane potential. Under identical conditions, the formation of reactive oxygen species (ROS) revealed with a general probe was significant at days 3 and 10 of differentiation and bearly detectable at day 6. NADPH oxidase (NOX)-2 activity determined with an immunocytochemical approach followed a very similar pattern. There was no evidence of mitochondrial ROS (mROS), as detected with a selective fluorescence probe, at days 3 and 6, possibly due to the triggering of the Nrf-2 antioxidant response. mROS were instead clearly detected at day 10, concomitantly with the accumulation of very large LDs, oxidation of both cardiolipin and thioredoxin 2, and decreased mitochondrial glutathione. In conclusion, the morphological and biochemical changes of differentiating SW872 cells are accompanied by the discontinuous formation of ROS derived from NOX-2, increasingly implicated in adipogenesis and adipose tissue dysfunction. In addition, mROS formation was significant only in the late phase of differentiation and was associated with mitochondrial dysfunction.

Arsenite impinges on endoplasmic reticulum-mitochondria crosstalk to elicit mitochondrial ROS formation and downstream toxicity.

Arsenite is an important carcinogen and toxic compound, causing various deleterious effects through multiple mechanisms. In this review, we focused on mitochondrial ROS (mitoROS) and discussed on the mechanisms mediating their formation. The metalloid promotes direct effects in mitochondria, resulting in superoxide formation only under conditions of increased mitochondrial Ca2+ concentration ([Ca2+]m). In this perspective, the time of exposure and concentration requirements for arsenite were largely conditioned by other effects of the metalloid in specific sites of the endoplasmic reticulum (ER). Arsenite induced a slow and limited mobilization of Ca2+ from IP3R via a saturable mechanism, failing to increase the [Ca2+]m. This effect was however associated with the triggering of an intraluminal crosstalk between the IP3R and the ryanodine receptor (RyR), causing a large and concentration dependent release of Ca2+ from RyR and a parallel increase in [Ca2+]m. Thus, the Ca2+-dependent mitoO2-. formation appears to be conditioned by the spatial/functional organization of the ER/mitochondria network and RyR expression. We also speculate on the possibility that the ER stress response might regulate the above effects on the intraluminal crosstalk between the IP3R and the RyR via oxidation of critical thiols mediated by the H2O2 locally released by oxidoreductin 1α.

Mitochondrial reactive oxygen species: the effects of mitochondrial ascorbic acid vs untargeted and mitochondria-targeted antioxidants.

PREMISE: Mitochondria represent critical sites for reactive oxygen species (ROS) production, which dependent on concentration is responsible for the regulation of both physiological and pathological processes. PURPOSE: Antioxidants in mitochondria regulate the redox balance, prevent mitochondrial damage and dysfunction and maintain a physiological ROS-dependent signaling. The aim of the present review is to provide critical elements for addressing this issue in the context of various pharmacological approaches using antioxidants targeted or non-targeted to mitochondria. Furthermore, this review focuses on the mitochondrial antioxidant effects of ascorbic acid (AA), providing clues on the complexities associated with the cellular uptake and subcellular distribution of the vitamin. CONCLUSIONS: Antioxidants that are not specifically targeted to mitochondria fail to accumulate in significant amounts in critical sites of mitochondrial ROS production and may eventually interfere with the ensuing physiological signaling. Mitochondria-targeted antioxidants are more effective, but are expected to interfere with the mitochondrial ROS-dependent physiologic signaling. AA promotes multiple beneficial effects in mitochondria. The complex regulation of vitamin C uptake in these organelles likely contributes to its versatile antioxidant response, thereby providing a central role to the vitamin for adequate control of mitochondrial dysfunction associated with increased mitochondrial ROS production.

SVCT2-Dependent plasma and mitochondrial membrane transport of ascorbic acid in differentiating myoblasts.

The Na+-dependent Vitamin C transporter 2 (SVCT2) is expressed in the plasma and mitochondrial membranes of various cell types. This notion was also established in proliferating C2C12 myoblasts (Mb), in which the transporter was characterised by a high and low affinity in the plasma and mitochondrial membranes, respectively. In addition, the mitochondrial expression of SVCT2 appeared particularly elevated and, consistently, a brief pre-exposure to low concentrations of Ascorbic Acid (AA) abolished mitochondrial superoxide formation selectively induced by the cocktail arsenite/ATP. Early myotubes (Mt) derived from these cells after 4 days of differentiation presented evidence of slightly increased SVCT2 expression, and were characterised by kinetic parameters for plasma membrane transport of AA in line with those detected in Mb. Confocal microscopy studies indicated that the mitochondrial expression of SVCT2 is well preserved in Mt with one or two nuclei, but progressively reduced in Mt with three or more nuclei. Cellular and mitochondrial expression of SVCT2 was found reduced in day 7 Mt. While the uptake studies were compromised by the poor purity of the mitochondrial preparations obtained from day 4 Mt, we nevertheless obtained evidence of poor transport of the vitamin using the same functional studies successfully employed with Mb. Indeed, even greater concentrations of/longer pre-exposure to AA failed to induce scavenging of mitochondrial superoxide in Mt. These results are therefore indicative of a severely reduced mitochondrial uptake of the vitamin in early Mt, attributable to decreased expression as well as impaired activity of mitochondrial SVCT2.

Arsenite-Induced Mitochondrial Superoxide Formation: Time and Concentration Requirements for the Effects of the Metalloid on the Endoplasmic Reticulum and Mitochondria.

The present study used human myeloid leukemia U937 cells, a versatile promonocytic cellular system that, based on its endoplasmic reticulum (ER)/mitochondria functional relationships, responds to low micromolar concentrations of arsenite with a single, defined mechanism of superoxide (O2 -.) formation. Under these conditions, we observe an initial Ca2+ mobilization from the ER associated with the mitochondrial accumulation of the cation, which is followed by Ca2+-dependent mitochondrial O2 -. (mitoO2 -.) formation. These events, which were barely detectable after 3 hours, were better appreciated at 6 hours. We found that markedly shorter exposure to arsenite and lower concentrations of arsenite are required to induce extensive O2 - formation in cells supplemented with inositol-1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR) agonists. Indeed, nanomolar arsenite induced maximal O2 -. formation after only 10 minutes of exposure, and this response was uniquely dependent on the enforced mitochondrial Ca2+ accumulation. The dramatic anticipation of and sensitization to the effects of arsenite caused by the IP3R or RyR agonists were accompanied by a parallel significant genotoxic response in the absence of detectable mitochondrial dysfunction and cytotoxicity. We conclude that the prolonged, low-micromolar arsenite exposure paradigm resulting in mitoO2 -. formation is necessary to affect Ca2+ homeostasis and accumulate the cation in mitochondria. The arsenite requirements to promote mitoO2 -. formation in the presence of sufficient mitochondrial Ca2+ were instead remarkably lower in terms of both concentration and time of exposure. These conditions were associated with the induction of extensive DNA strand scission in the absence of detectable signs of toxicity. SIGNIFICANCE STATEMENT: In respiration-proficient cells, arsenite causes mitochondrial Ca2+ accumulation and Ca2+-dependent mitochondrial superoxide formation. We now report that the second event requires remarkably lower concentrations of and time of exposure to the metalloid than the former. Indeed, a brief exposure to nanomolar levels of arsenite produced maximal effects under conditions in which the mitochondrial Ca2+ concentration ([Ca2+]m) was increased by inositol-1,4,5-trisphosphate receptor or ryanodine receptor agonists. Hence, specific substances or conditions enhancing the [Ca2+]m may potentiate the deleterious effects of arsenite by selectively increasing mitochondrial superoxide formation.

The compartmentalised nature of the mechanisms governing superoxide formation and scavenging in cells exposed to arsenite.

In this study, respiration-proficient (RP) and -deficient (RD) cells were exposed to 2.5 or 10 µM arsenite to generate superoxide (O2-.) respectively in the mitochondrial respiratory chain or via NADPH oxidase activation. These treatments, while causing similar, although mitochondrial permeability transition-dependent (RP-cells) or independent (RD-cells), delayed apoptosis, surprisingly generated identical kinetics and levels of dihydrorhodamine oxidation, indicative of O2-. formation. These similarities were attributable to the involvement of a common upstream event resulting in activation of the two O2-.-generating systems, and to intrinsic features of the cells. Both mechanisms required an initial and sequential mobilization of Ca2+ from the inositol-1,4,5-trisphosphate receptor and the ryanodine receptor (RyR), with however different implications. The close contacts existing between the RyR and the mitochondria created optimal conditions for the Ca2+ clearance, and the ensuing formation of O2-. in RP-cell mitochondria. Exposure to low concentrations of l-ascorbic acid (AA) transported by high affinity mechanisms in cells and mitochondria, suppressed O2-. formation. Much more Ca2+, and hence more arsenite, was necessary to promote NADPH oxidase activation in RD-cells, as a consequence of the cytosolic dilution and mitochondrial clearance of Ca2+. For the same reasons, an exposure to high concentrations of AA was required to suppress O2-. formation under these conditions.

Prostaglandin E2 Signals Through E Prostanoid Receptor 2 to Inhibit Mitochondrial Superoxide Formation and the Ensuing Downstream Cytotoxic and Genotoxic Effects Induced by Arsenite.

We investigated the effects of prostaglandin E2 (PGE2), an important inflammatory lipid mediator, on the cytotoxicity-genotoxicity induced by arsenite. With the use of a toxicity paradigm in which the metalloid uniquely induces mitochondrial superoxide (mitoO2 -.) formation, PGE2 promoted conditions favoring the cytosolic accumulation of Bad and Bax and abolished mitochondrial permeability transition (MPT) and the ensuing lethal response through an E prostanoid receptor 2/adenylyl cyclase/protein kinase A (PKA) dependent signaling. It was, however, interesting to observe that, under the same conditions, PGE2 also abolished the DNA-damaging effects of arsenite and that this response was associated with an unexpected suppression of mitoO2 -. formation. We conclude that PGE2 promotes PKA-dependent inhibition of mitoO2 -. formation, thereby blunting the downstream responses mediated by these species, leading to DNA strand scission and MPT-dependent apoptosis. These findings are therefore consistent with the possibility that, in cells responding to arsenite with mitoO2 -. formation, PGE2 fails to enhance-but rather decreases-the risk of neoplastic transformation associated with genotoxic events.

Calcium signals between the ryanodine receptor- and mitochondria critically regulate the effects of arsenite on mitochondrial superoxide formation and on the ensuing survival vs apoptotic signaling.

A low concentration of arsenite (6 h), selectively stimulating the intraluminal crosstalk between the inositol-1, 4, 5-triphosphate receptor and the ryanodine receptor (RyR), increased the mitochondrial transport of RyR-derived Ca2+ through the mitochondrial Ca2+ uniporter. This event was characterized in intact and permeabilized cells, and was shown to be critical for mitochondrial superoxide (mitoO2.-) formation. Inhibition of mitochondrial Ca2+ accumulation therefore prevented the effects of arsenite, in both the mitochondrial (e.g., cardiolipin oxidation) and extramitochondrial (e.g., DNA single- strand breakage) compartments, and suppressed the Nrf2/GSH survival signaling. The effects of arsenite on Ca2+ homeostasis and mitoO2.- formation were reversible, as determined after an additional 10 h incubation in fresh culture medium and by measuring long-term viability. A 16 h continuous exposure to arsenite instead produced a sustained increase in the cytosolic and mitochondrial Ca2+ concentrations, a further increased mitoO2.- formation and mitochondrial permeability transition. These events, followed by delayed apoptosis (48 h), were sensitive to treatments/manipulations preventing mitochondrial Ca2+ accumulation. Interestingly, cells remained viable under conditions in which the deregulated Ca2+ homeostasis was not accompanied by mitoO2.-formation. In conclusion, we report that the fraction of Ca2+ taken up by the mitochondria in response to arsenite derives from the RyR. Mitochondrial Ca2+ appears critical for mitoO2.- formation and for the triggering of both the cytoprotective and apoptotic signaling. The effects of arsenite were reversible, whereas its prolonged exposure caused a sustained increase in mitochondrial Ca2+ and mitoO2.- formation, and the prevalence of the apoptotic vs survival signaling.

The In Vitro Activity of Angelica archangelica L. Essential Oil on Inflammation.

The use of herbs with medicinal value and biomedical effects has increased tremendously in the last years. However, inadequate basic knowledge of their mode of action is the main issue related to phytotherapy, although they have shown promising potential. To provide insights into these important issues, we tested here on appropriate in vitro models the efficacy of Angelica archangelica essential oil (Aa-EO) for anti-inflammatory properties. The results demonstrated that Aa-EO induced significant apoptosis and necrosis at high doses in U937 cells. We used nontoxic concentrations to treat for anti-inflammatory capacity. The results also demonstrated a decreased proinflammatory cytokine interleukin-6 level in human umbilical vein endothelial cells, as senescence in vitro model, when cells are challenged with lipopolysaccharide (LPS), one of the most powerful proinflammatory inducer in the presence of Aa-EO. In addition, down expression of miR-126 and miR-146a (inflammamirs) produced by LPS stimulation was reverted by Aa-EO simultaneous treatment. These results provide noteworthy basis for the development/formulation of new drugs for future clinical uses and new food products or dietary supplements for contrasting inflammation.

Melatonin protects hippocampal HT22 cells from the effects of serum deprivation specifically targeting mitochondria.

Neurons contain a high number of mitochondria, these neuronal cells produce elevated levels of oxidative stress and live for a long time without proliferation; therefore, mitochondrial homeostasis is crucial to their health. Investigations have recently focused on mitochondrial dynamics revealing the ability of these organelles to change their distribution and morphology. It is known that mitochondrial fission is necessary for the transmission of mitochondria to daughter cells during mitosis and mitochondrial fragmentation has been used as an indicator of cell death and mitochondrial dysfunction. Oxidative stress is a trigger able to induce changes in the mitochondrial network. The aim of the present study was to determine the effects of melatonin on the mitochondrial network in HT22 serum-deprived cells. Our results showed that serum deprivation increased reactive oxygen species (ROS) content, promoted the activation of plasma membrane voltage-dependent anion channels (VDACs) and affected the expression of pDRP1 and DRP1 fission proteins. Moreover, parallel increases in apoptotic and autophagic features were found. Damaged and dysfunctional mitochondria are deleterious to the cell; hence, the degradation of such mitochondria through mitophagy is crucial to cell survival. Our results suggest that melatonin supplementation reduces cell death and restores mitochondrial function through the regulation of autophagy.

Low Concentrations of Arsenite Target the Intraluminal Inositol 1, 4, 5-Trisphosphate Receptor/Ryanodine Receptor Crosstalk to Significantly Elevate Intracellular Ca2.

Arsenite is an established human carcinogen that induces cytotoxic and genotoxic effects through poorly defined mechanisms involving the formation of reactive oxygen species (ROS) and deregulated Ca2+ homeostasis. We used variants of the U937 cell line to address the central issue of the mechanism whereby arsenite affects Ca2+ homeostasis. We found that 6-hour exposure to the metalloid (2.5 µM), although not associated with an immediate or delayed toxicity, causes a significant increase in the intracellular Ca2+ concentration ([Ca2+]i) through a mechanism characterized by the following components: 1) it was not affected by ROS produced under the same conditions; 2) a small amount of Ca2+ was mobilized from the inositol-1,4,5-trisphosphate receptor (IP3R), and this response was not augmented by greater concentrations of the metalloid; 3) large amounts of Ca2+ were instead dose dependently mobilized from the ryanodine receptor (RyR) in response to IP3R stimulation; 4) the cells maintained an intact responsiveness to agonist-stimulated Ca2+ mobilization from both channels; 5) arsenite, even at 5-10 µM, failed to directly mobilize Ca2+ from the RyR; and 6) arsenite failed to enhance Ca2+ release from the RyR under conditions in which the [Ca2+]i was increased by either RyR agonists or ionophore-stimulated Ca2+ uptake. We therefore conclude that arsenite elevates the [Ca2+]i by directly targeting the IP3R and its intraluminal crosstalk with the RyR. This mechanism likely mediates mitochondrial superoxide formation, downstream damage on various biomolecules (including genomic DNA), and mitochondrial dysfunction/apoptosis eventually occurring after longer incubation to, or exposure to greater concentrations of, arsenite.