Search Results (6)

Objective: Joint injury precipitates post-traumatic osteoarthritis (PTOA) via chondrocyte mitochondrial oxidative damage. Carbon monoxide (CO) is a small molecule with potent antioxidant and mitochondrial benefits in other tissues that have not been explored in healthy chondrocytes. We hypothesized that CO would subvert the mitochondrial effects of articular cartilage injuries upon resident chondrocytes. Design: We evaluated intra-articular delivery of a novel carbon monoxide-containing foam (COF). We used in vitro impact injuries to explore mitochondrial and redox endpoints after CO exposure. We then applied intra-articular injections of COF or control room air foam (RAF) to assess safety, efficacy, and other intra-articular responses. Results: COF increased the expression of HO1 and mitofusin-1 within 1 h and this increase was sustained for 12 h in vitro. COF increased chondrocyte mitochondrial respiration by 40% and increased reduced (not oxidized) thiols by 50% following in vitro injury to osteochondral explants. After cartilage injury, COF prevented the formation of 3-nitrotyrosine and the loss of articular chondrocyte mitochondria. When injected intra-articularly, COF was retained for 24 h post-injection in mouse stifle joints. It increased HO1 in those joints, enhanced reduced thiol levels in rabbit stifle joints, and exhibited no toxicity 1 and 4 weeks after injection. Conclusions: This study supports the hypothesis that CO functions as an antioxidant for articular chondrocytes by supporting mitochondria and intracellular GSH in the presence or absence of cartilage injury. Challenges in delivering exogenous CO have limited its preclinical development, but new CO-releasing materials like COF may enable new examinations of this promising small molecule. Full article

Fucoxanthin is the most abundant carotenoid found in marine brown algae that exhibits several healthy properties. Dietary fucoxanthin is metabolized in the intestine, plasma, and other tissues to various metabolites, including fucoxanthinol. In this regard, the contribution of fucoxanthinol to the healthy properties of its precursor, fucoxanthin, against pathogenetic events associated with neurodegenerative diseases remains unexplored. Here, we evaluated and compared the antioxidant and neuroprotective effects of the carotenoids fucoxanthin and fucoxanthinol in in vitro models of Alzheimer’s (AD) and Parkinson’s (PD) disease. Neuronal SH-SY5Y cells were used to evaluate the antioxidant properties of the carotenoids against ABTS radical in the membrane and cytoplasm and oxidative stress elicited by tert-butyl hydroperoxide using the 2′,7′-dichlorodihydrofluorescein diacetate probe. We also assessed the ability of the carotenoids to increase the glutathione (GSH) and activate the Nrf2/Keap1/ARE pathway using the monochlorobimane probe and western blotting method, respectively. The neuroprotective effects of the carotenoids against the neurotoxicity generated by oligomers of Beta-Amyloid (1–42) peptide (OAβ) and 6-hydroxydopamine (6-OHDA), which are neurotoxins of AD and PD, respectively, were finally evaluated in the same neuronal cells using the thiazolyl blue tetrazolium bromide assay. Both carotenoids could reach the cytoplasm, which explains the mainly free radical scavenging activity at this level. Notably, fucoxanthinol had higher and lower antioxidant activity than fucoxanthin at extracellular and cellular levels. Although studied carotenoids exerted the ability to activate the Nrf2/Keap1/ARE pathway, leading to an increase of intracellular GSH, our results suggested that the antioxidant activity of the carotenoids could be mainly attributed to their radical scavenging activity in neuronal membrane and cytoplasm, where they accumulate. Fucoxanthinol also shared similar neuroprotective effects as fucoxanthin against the neurotoxicity generated by OAβ and 6-OHDA, suggesting a potential neuroprotective contribution to the action of fucoxanthin administered as a food supplement in in vivo experimental models. These results encourage further research to evaluate the bioavailability of fucoxanthinol and other metabolites of fucoxanthin at the brain level to elucidate the dietary neuroprotective potential of fucoxanthin. Full article

Thiol compounds including predominantly glutathione (GSH) are key components of redox homeostasis, which are involved in the protection and regulation of mammalian cells. The assessment of cell redox status by means of in situ analysis of GSH in living cells is often preferable over established assays in cell lysates due to fluctuations of the GSH pool. For this purpose, we propose a microplate assay with monochlorobimane (MCB) as an available fluorescent probe for GSH, although poorly detected in the microplate format. In addition to the new procedure for improved MCB-assisted GSH detection in plate-grown cells and its verification with GSH modulators, this study provides a useful methodology for the evaluation of cell redox status probed through relative GSH content and responsiveness to both supplemented thiols and variation in oxygen pressure. The roles of extracellular interactions of thiols and natural variability of cellular glutathione on the assay performance were emphasized and discussed. The results are of broad interest in cell biology research and should be particularly useful for the characterization of pathological cells with decreased GSH status and increased oxidative status as well as redox-modulating factors. Full article

Colorectal cancer (CRC) is on the rise in industrialized countries, which is why it is important to find new compounds that are effective, with little or no adverse health effects. CRC arises from some cells of the epithelium which, following a series of genetic or epigenetic mutations, obtain a selective advantage. This work consists of a review on endogenous and exogenous antioxidant products that may have an efficacy in the treatment of CRC and an experimental study, in which the treatment was carried out with a natural compound with antitumor and antiproliferative activity, Prunus spinosa Trigno ecotype, patented by us, on HCT116 colorectal carcinoma cell line. The superoxide content was quantified after the treatments at different concentrations (2, 5, or 10 mg/mL) by means of the DHR123 probe; loss of the mitochondrial membrane potential with the tetramethylrodamine methyl ester (TMRM) cationic probe and reduced glutathione content (GSH) from monochlorobimane (MCB). This study revealed the importance of a careful choice of the concentration of the natural compound to be used in the CRC, due to the presence of a paradoxical effect, both antioxidant and pro-oxidant, depending on the different physiological conditions of the cell. Full article

In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes. Full article

Cell-permeable fluorescent chemosensors (calcein, monochlorobimane, and a recently reported spiropyran-based sensor SP2) have been incorporated into yeast total lipid extract-based liposomes to suppress inherent cell permeability to allow the detection of extracellular Ca²⁺, GSH, and Zn²⁺, respectively. The repurposed sensors have enhanced aqueous solubility and the ability to quantitatively measure biologically relevant concentrations of Ca²⁺ (0.25 mM–1 mM), Zn²⁺ (6.25 µM–50 µM), and GSH (0.25 mM–1 mM) by fluorescence in aqueous media. In addition, the liposomal sensors are nontoxic to HEK293 cells and have the ability to detect exogenously added Zn²⁺ (1 mM), Ca²⁺ (1 mM), or GSH (1 mM) near cells without internalisation. This new sensing platform provides a means to repurpose a range of intracellular fluorescent sensors to specifically detect extracellular analytes, while also improving biocompatibility for overall enhanced use in a wide range of biomedical applications. Full article