New Biocompatible ß-Phosphorylated Linear Nitrones Targeting Mitochondria: Protective Effect in Apoptotic Cells.

The mitochondrion, an essential organelle involved in cellular respiration, energy production, and cell death, is the main cellular source of reactive oxygen species (ROS), including superoxide. Mitochondrial diseases resulting from uncontrolled/excess ROS generation are an emerging public health concern and there is current interest in specific mitochondriotropic probes to get information on in-situ ROS production. As such, nitrones vectorized by the triphenylphosphonium (TPP) cation have recently drawn attention despite reported cytotoxicity. Herein, we describe the synthesis of 13 low-toxic derivatives of N-benzylidene-1-diethoxyphosphoryl-1-methylethylamine N-oxide (PPN) alkyl chain-grafted to a pyridinium, triethylammonium or berberinium lipophilic cation. These nitrones showed in-vitro superoxide quenching activity and EPR/spin-trapping efficiency towards biologically relevant free radicals, including superoxide and hydroxyl radicals. Their mitochondrial penetration was confirmed by 31 P NMR spectroscopy, and their anti-apoptotic properties were assessed in Schwann cells treated with hydrogen peroxide. Two pyridinium-substituted PPNs were identified as potentially better alternatives to TPP nitrones conjugates for studying mitochondrial oxidative damage.

Novel Sterically Crowded and Conformationally Constrained α-Aminophosphonates with a Near-Neutral pKa as Highly Accurate 31P NMR pH Probes. Application to Subtle pH Gradients Determination in Dictyostelium discoideum Cells.

In order to discover new 31P NMR markers for probing subtle pH changes (<0.2 pH unit) in biological environments, fifteen new conformationally constrained or sterically hindered α-aminophosphonates derived from diethyl(2-methylpyrrolidin-2-yl)phosphonate were synthesized and tested for their pH reporting and cytotoxic properties in vitro. All compounds showed near-neutral pKas (ranging 6.28−6.97), chemical shifts not overlapping those of phosphorus metabolites, and spectroscopic sensitivities (i.e., chemical shifts variation Δδab between the acidic and basic forms) ranging from 9.2−10.7 ppm, being fourfold larger than conventional endogenous markers such as inorganic phosphate. X-ray crystallographic studies combined with predictive empirical relationships and ab initio calculations addressed the inductive and stereochemical effects of substituents linked to the protonated amine function. Satisfactory correlations were established between pKas and both the 2D structure and pyramidalization at phosphorus, showing that steric crowding around the phosphorus is crucial for modulating Δδab. Finally, the hit 31P NMR pH probe 1b bearing an unsubstituted 1,3,2-dioxaphosphorinane ring, which is moderately lipophilic, nontoxic on A549 and NHLF cells, and showing pKa = 6.45 with Δδab = 10.64 ppm, allowed the first clear-cut evidence of trans-sarcolemmal pH gradients in normoxic Dictyostelium discoideum cells with an accuracy of <0.05 pH units.

New Amino-Acid-Based ß-Phosphorylated Nitroxides for Probing Acidic pH in Biological Systems by EPR Spectroscopy.

There is increasing interest in measuring pH in biological samples by using nitroxides with pH-dependent electron paramagnetic resonance (EPR) spectra. Aiming to improve the spectral sensitivity (ΔaX ) of these probes (i.e., the difference between the EPR hyperfine splitting (hfs) in their protonated and unprotonated forms), we characterized a series of novel linear α-carboxy, α'-diethoxyphosphoryl nitroxides constructed on an amino acid core and featuring an (α or α')-C-H bond. In buffer, the three main hfs (aN , aH , and aP ) of their EPR spectra vary reversibly with pH and, from aP or aH titration curves, a two- to fourfold increase in sensitivity was achieved compared to reference imidazoline or imidazolidine nitroxides. The crystallized carboxylate 10 b (pKa ≈3.6), which demonstrated low cytotoxicity and good resistance to bioreduction, was applied to probe stomach acidity in rats. The results pave the way to a novel generation of highly sensitive EPR pH markers.

Improving the Antioxidant Properties of Calophyllum inophyllum Seed Oil from French Polynesia: Development and Biological Applications of Resinous Ethanol-Soluble Extracts.

Tamanu oil from Calophyllum inophyllum L. has long been used in traditional medicine. Ethanol extraction was found the best strategy for recovering bioactive compounds from the resin part of Tamanu oil, yielding two neutral and acidic resins fractions with high phenolics, flavonoids and pyranocoumarins concentrations. A further cascade of LPLC/HPLC separations of neutral and acidic resin fractions allowed identifying fifteen metabolites, and among them, calanolide D and 12-oxocalanolide A (both in neutral fraction) were first identified from a natural source. All these extracts, subfractions and isolated metabolites demonstrated increased free radical scavenging, antioxidant, anti-inflammatory, antimicrobial and antimycobacterial activity compared to Tamanu oil and its de-resinated lipid phase. Overall, these results could promote resinous ethanol-soluble Tamanu oil extracts as a useful multifaceted and renewable medicinal resource.

Inhibitory effect of fungoid chitosan in the generation of aldehydes relevant to photooxidative decay in a sulphite-free white wine.

The reaction pathways were investigated by which a fungoid chitosan (CsG) may protect against photooxidative decay of model solutions and a sulphite-free white wine. Samples containing CsG were dark incubated for 2 days before exposure to fluorescent lighting for up to 21 days in the presence of wine like (+)-catechin and/or iron doses. In both systems CsG at winemaking doses significantly reduced the photoproduction of acetaldehyde and, to a better extent, glyoxylic acid, two key reactive aldehydes implicated in wine oxidative spoilage. After 21 days, CsG was two-fold more effective than sulphur dioxide in preventing glyoxylic acid formation and minimizing the browning of white wine. Among the antioxidant mechanisms involved in CsG protective effect, iron chelation, and hydrogen peroxide quenching were demonstrated. Besides, the previously unreported tartrate displacement from the [iron(III)-tartrate] complex was revealed as an additional inhibitory mechanism of CsG under photo-Fenton oxidation conditions.

Chitosan as an antioxidant alternative to sulphites in oenology: EPR investigation of inhibitory mechanisms.

The efficacy against oxidative degradation in model and sulphite-free white wines of two commercial, insoluble chitosans (one being approved for winemaking) were investigated by electron paramagnetic resonance (EPR). Both compounds at various doses significantly inhibited the formation of α-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN)-1-hydroxyethyl adducts under normal wine storage conditions. Pre-incubation with 2 g/L chitosan followed by filtration had a better effect than adding 50 mg/L sulphur dioxide to the experimental Chardonnay wine on the release of 4-POBN adducts after 6 days of incubation with 100 µM iron(II). In a relevant photooxidative system acetaldehyde formation was significantly reduced after 6 days of incubation. Parallel EPR tests were performed to assess the importance of metal chelation (iron and copper) versus direct scavenging of hydroxyl radicals on the effect of chitosan. The present data support the potentiality of using biocompatible chitosan as a healthier complement and/or alternative to sulphur dioxide against white wine oxidative spoilage.

A fluorescent homogeneous assay for myeloperoxidase measurement in biological samples. A positive correlation between myeloperoxidase-generated HOCl level and oxidative status in STZ-diabetic rats.

Myeloperoxidase (MPO) is a key enzyme derived from leukocytes which is associated with the initiation and progression of many inflammatory diseases. Increased levels of MPO may contribute to cellular dysfunction and tissues injury by producing highly reactive oxidants such as hypochlorous acid (HOCl). Myeloperoxidase-generated HOCl is therefore considered as a relevant biomarker of oxidative stress-related damage and its quantitation is of great importance to the study of disease progression. In this context, the current study describes a rapid, sensitive and homogeneous fluorescence-based method for detecting the MPO chlorination activity in biological samples. This assay utilizes 7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde oxime as a selective probe for HOCl detection, and is adapted to 96-well microplates to allow high-throughput quantitation of active MPO. The ability of the method to monitor HOCl release was further investigated in hyperglycemic streptozotocin-treated diabetic rats. The data proved that the present assay has a reliable performance when quantitating the active MPO in the plasma of diabetic animals, a feature of inflammatory disease found concomitant with an elevation of protein carbonyls levels and lipid peroxidation and which was negatively correlated with the ratio of reduced-to-oxidized glutathione.

Antidiabetic, antioxidant and anti inflammatory properties of water and n-butanol soluble extracts from Saharian Anvillea radiata in high-fat-diet fed mice.

ETHNOPHARMACOLOGICAL RELEVANCE: According to Saharian traditional medicine, Anvillea radiata Coss. & Dur. (Asteraceae) has been valued for treating a variety of ailments such as gastro-intestinal, liver and pulmonary diseases, and has gained awareness for its beneficial effect on postprandial hyperglycemia. However, to best of our knowledge, no detailed study of the antidiabetic curative effects of this plant has been conducted yet. AIM OF THE STUDY: To determine the hypoglycemic and antidiabetic effect of dietary supplementation with Anvillea radiata extracts on high-fat-diet (HFD)-induced obesity and insulin resistance in C57BL/6J mice in relation with antioxidant, anti-inflammatory, pancreatic beta-cells and skeletal muscle protection, and digestive enzyme inhibiting properties. MATERIALS AND METHODS: Six extracts (water soluble and organic) from aerial parts of the plant were analyzed phytochemically (total phenolic and flavonoid content) and screened for in vitro superoxide (by chemiluminescence) and hydroxyl radical (by electron paramagnetic resonance spin-trapping) scavenging, antioxidant (DPPH, TRAP and ORAC assays), xanthine oxidase, metal chelating, α-amylase and α-glucosidase inhibitory property, and protective effects on copper-induced lipoprotein oxidation. Then selected hydroalcoholic and aqueous extracts were assessed for toxicity in normal human lung fibroblasts and A549 cancer cells using FMCA and MTT assays. Two water-soluble extracts having the best overall properties were assessed for their (i) protective effect at 1-15µg/mL on metabolic activity of rat insulinoma-derived INS-1 cells exposed to hyperglycemic medium, and (ii) acute hypoglycemic effect on 16-weeks HFD-induced diabetic mice. Then diabetic mice were administered HFD supplemented by extracts (up to 150mg/kg/day) for 12 additional weeks using standard diet as control and the antidiabetic drug, metformin (150mg/kg), as positive control. Then the antidiabetic, anti-inflammatory and antioxidant activity of extracts were determined. RESULTS: Of the highly efficient polyphenolics-enriched hydroalcoholic and ethyl acetate extracts, the lyophilized aqueous (AQL) and butanol extracts were not toxic in cells (≤ 400µg/mL) or when given orally in normal mice (≤ 2000mg/kg), exerted a dose-dependent hypoglycemic action in diabetic mice, which was maximal at the dose of 150mg/kg. Upon administering this dose for 12 weeks, both extracts significantly ameliorated body weight control capacity, recovery of plasma glucose and insulin level, reduced oxidative stress in blood, myocardial and skeletal muscles, and improved hyperlipidemic and inflammatory status. Moreover, diabetes-related complications were optimally ameliorated by oral therapy based on halved doses (75mg/kg) of a mixture of AQL and metformin. CONCLUSIONS: Current investigation supports the traditional medicinal usage of Anvillea radiata and suggests that both readily accessible and low-cost bio-extracts have the potency to develop an antihyperglycemic, antihyperlipidemic and protective agent against beta-cells and muscle dysfunction at doses compatible with the common practices of indigenous people for the management of metabolic disorders.

On the vasoprotective mechanisms underlying novel ß-phosphorylated nitrones: Focus on free radical characterization, scavenging and NO-donation in a biological model of oxidative stress.

A series of new hybrid 2-(diethoxyphosphoryl)-N-(benzylidene)propan-2-amine oxide derivatives with different aromatic substitution (PPNs) were synthesized. These molecules were evaluated for their EPR spin trapping potential on eleven different radicals and NO-donation properties in vitro, cytotoxicity and vasoprotective effect on precontracted rat aortic rings. A subfamily of the new PPNs featured an antioxidant moiety occurring in natural phenolic acids. From the experimental screening of these hydroxyphenyl- and methoxyphenyl-substituted PPNs, biocompatible nitrones 4d, and 4g-4i deriving from caffeic, gallic, ferulic and sinapic acids, which combined improved EPR probing of ROS formation, vasorelaxant action and antioxidant potency, might be potential drug candidate alternatives to PBN and its analogues.

DNA damage and oxidative stress induced by CeO2 nanoparticles in human dermal fibroblasts: Evidence of a clastogenic effect as a mechanism of genotoxicity.

The broad range of applications of cerium oxide (CeO2) nanoparticles (nano-CeO2) has attracted industrial interest, resulting in greater exposures to humans and environmental systems in the coming years. Their health effects and potential biological impacts need to be determined for risk assessment. The aims of this study were to gain insights into the molecular mechanisms underlying the genotoxic effects of nano-CeO2 in relation with their physicochemical properties. Primary human dermal fibroblasts were exposed to environmentally relevant doses of nano-CeO2 (mean diameter, 7 nm; dose range, 6 × 10(-5)-6 × 10(-3) g/l corresponding to a concentration range of 0.22-22 µM) and DNA damages at the chromosome level were evaluated by genetic toxicology tests and compared to that induced in cells exposed to micro-CeO2 particles (mean diameter, 320 nm) under the same conditions. For this purpose, cytokinesis-blocked micronucleus assay in association with immunofluorescence staining of centromere protein A in micronuclei were used to distinguish between induction of structural or numerical chromosome changes (i.e. clastogenicity or aneuploidy). The results provide the first evidence of a genotoxic effect of nano-CeO2, (while not significant with micro-CeO2) by a clastogenic mechanism. The implication of oxidative mechanisms in this genotoxic effect was investigated by (i) assessing the impact of catalase, a hydrogen peroxide inhibitor, and (ii) by measuring lipid peroxidation and glutathione status and their reversal by application of N-acetylcysteine, a precusor of glutathione synthesis in cells. The data are consistent with the implication of free radical-related mechanisms in the nano-CeO2-induced clastogenic effect, that can be modulated by inhibition of cellular hydrogen peroxide release.