Redox Imbalance In Cells Research Articles

Single fluorescent probe reveals glutathione-viscosity crosstalk in the endoplasmic reticulum during ferroptosis and extracellular vesicles in synergistic ferroptosis-apoptosis

Ferroptosis is an iron-dependent cell death triggered by lipid peroxidation accumulation, representing a redox imbalance in cells. The increased knowledge of ferroptosis has led to various potential strategies to develop new cancer therapies, such as synergistic ferroptosis-apoptosis. The endoplasmic reticulum (ER) is the major hub for lipid metabolism in cells and plays a central role in lipid peroxidation. Glutathione (GSH) and viscosity levels in ER are important parameters for intracellular redox homeostasis, which remain unclear in ferroptosis and synergistic ferroptosis-apoptosis. This study developed an ER-targeting fluorescent probe (MMPY) with a dual-emission response to GSH and viscosity. Fluorescent cell imaging using MMPY revealed different GSH-viscosity crosstalk in the ER for the four ferroptosis pathways induced by erastin, RSL3, FIN56, and FINO2. GSH and viscosity changes in the ER during synergistic ferroptosis-apoptosis were also investigated. Interestingly, the new types of extracellular vesicles were observed during the synergistic process, which burdened the ER fragments with different GSH contents.

Evaluation of nanoplastics-induced redox imbalance in cells, larval zebrafish, and daphnia magna with a superoxide anion radical fluorescent probe

Nanoplastics (NPs) is a novel plastic contaminant that could be taken up by cells and lead to severe biotoxicity toxicity, NPs in cells can cause oxidant damage by inducing reactive oxygen species (ROS) production and lead to acute inflammation. As a major ROS which related to many kinds of physiological and pathological processes, superoxide anion radical (O2•−) could be utilized as a signal of oxidant damage effected by NPs exposure in vivo. To detect the toxic damage mechanism of NPs, a fluorescence probe Bcy-OTf has been developed to monitor O2•− fluctuations content in cells and aquatic organisms after exposure to NPs. The probe has a high sensitivity (LOD = 20 nM) and a rapid responsive time (within 6 min), and it has high selectivity and low cytotoxicity to analysis the levels of the endogenous O2•−. Endogenous O2•− induced by NPs in living cells, Daphnia magna and larval zebrafish were analyzed. Moreover, the results confirmed the key role of MAPK and NF-κB pathway in NPs stimulation mechanisms in cells. This study indicated that Bcy-OTf can precisely assess the fluctuations of endogenous O2•−, which has potential for applying in further analysis mechanisms of NPs biological risks.

Dithiothreitol reduces oxidative stress and necrosis caused by ultraviolet A radiation in L929 fibroblasts.

Ultraviolet A (UVA) radiation, present in sunlight, can induce cell redox imbalance leading to cellular damage and even cell death, compromising skin health. Here, we evaluated the in vitro antioxidant and photochemoprotective effect of dithiothreitol (DTT). DTT neutralized the free radicals 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+), 2,2-diphenyl-1-picrylhydrazyl (DPPH·), and superoxide anion (O2·-) in in vitro assays, as well as the ferric ion (Fe3+) in the ferric reducing antioxidant power (FRAP) assay. We also evaluated the effect of DTT pre-treatment in L929 dermal fibroblasts and DTT (50 and 100µM) led to greater cell viability following UVA-irradiation compared to cells that were untreated. Furthermore, the pre-treatment of cells with DTT prevented the increase of intracellular reactive oxygen species (ROS) production, including hydrogen peroxide (H2O2), lipid peroxidation, and DNA condensation, as well as the decrease in mitochondrial membrane potential (Δψm), that occurred following irradiation in untreated cells. The endogenous antioxidant system of cells was also improved in irradiated cells that were DTT pre-treated compared to the untreated cells, as the activity of the superoxide dismutase (SOD) and catalase (CAT) enzymes remained as high as non-irradiated cells, while the activity levels were depleted in the untreated irradiated cells. Furthermore, DTT reduced necrosis in UVA-irradiated fibroblasts. Together, these results showed that DTT may have promising use in the prevention of skin photoaging and photodamage induced by UVA, as it provided photochemoprotection against the harmful effects of this radiation, reducing oxidative stress and cell death, due mainly to its antioxidant capacity.

A multicolor reversible fluorescent probe for detecting SO2 and H2O2: Revealing redox imbalance in ischemia-reperfusion model

The redox homeostasis between hydrogen peroxide (H2O2) and sulfur dioxide (SO2) is necessary to maintain the normal physiological function of biological system. However, Due to the lack of fluorescent probes for the reversible dual detection of SO2 and H2O2, it is not clear which of the above signaling molecules predominates to cause redox state imbalance in biological systems. Herein, we designed a new muti-color reversible fluorescence probe Mito-BHY based on ICT mechanism. The probe has three different fluorescence signals (blue, green, and red) before and after reacting to SO2 and H2O2, and it can also reversibly detect SO2 and H2O2 through changes in the green and red fluorescence signals. The probe Mito-BHY has the characteristics of favorite response to SO2 and H2O2 with fast response speed, highly selectivity and biocompatibility, and can be used to monitor SO2 and H2O2 in the living cells and mice. Using Mito-BHY, we discovered that the content of H2O2 increased during cell redox imbalance and ischemia reperfusion in the mice, while the content of SO2 remained basically unchanged. This work will contribute to the understanding of the variation of SO2 and H2O2 levels, and is expected to provide an effective tool for pathological studies of redox imbalance.

Early responses of maize seedlings to Cu stress include sharp decreases in gibberellins and jasmonates in the root apex.

Copper (Cu) interferes with numerous biological functions in plants, including plant growth, which is partly governed by plant hormones. In the present study, Cu stress effect on the roots of pre-emerging maize seedlings in terms of growth, nutrient composition, protein modifications, and root hormone homeostasis was investigated, focusing on possible metabolic differences between the root apex and the rest of the root tissues. Significant decreases in root length and root biomass after 72h of Cu exposure (50 and 100μM CuCl2), accompanied by reductions in Ca, Mg, and P root contents, were found. Cu also generated cell redox imbalance in both root tissues and revealed by altered enzymatic and non-enzymatic antioxidant defenses. Oxidative stress was evidenced by an increased protein carbonylation level in both tissues. Copper also induced protein ubiquitylation and SUMOylation and affected 20S proteasome peptidase activities in both tissues. Drastic reductions in ABA, IAA, JA (both free and conjugated), GA3, and GA4 levels in the root apex were detected under Cu stress. Our results show that Cu exposure generated oxidative damage and altered root hormonal homeostasis, mainly at the root apex, leading to a strong root growth inhibition. Severe protein post-translational modifications upon Cu exposure occurred in both tissues, suggesting that even when hormonal adjustments to cope with Cu stress occurred mainly at the root apex, the entire root is compromised in the protein turnover that seems to be necessary to trigger and/or to sustain defense mechanisms against Cu toxicity.

Targeted Manganese doped silica nano GSH-cleaner for treatment of Liver Cancer by destroying the intracellular redox homeostasis.

Background: Glutathione (GSH), the primary antioxidant in cells, could fight against oxidative stress. Tumor cells display a higher GSH level than normal cells for coping with the hyperoxidative state, which meets the requirements of enhanced metabolism and vicious proliferation. Therefore, the consumption of GSH will lead to cell redox imbalance and impede life activities. Herein, targeted sorafenib (SFB) loaded manganese doped silica nanoparticle (FaPEG-MnMSN@SFB) was constructed, which could destroy the intracellular redox homeostasis by consuming GSH.Methods: In this study, MnMSN was prepared by an optimized one-pot Stober's method for loading SFB, and FaPEG chain was modified on the surface of MnMSN to achieve long circulation and targeted delivery. The anticancer efficacy and mechanism of the designed FaPEG-MnMSN@SFB were assessed both in vitro and in vivo.Results: FaPEG-MnMSN@SFB exhibited efficient antitumor activity by dual depleting intracellular GSH (the degradation of MnMSN would consume intracellular GSH and the SFB would inhibit the effect of Xc- transport system to inhibit GSH synthesis). Moreover, disruption of redox balance would lead to apoptosis and reactive oxygen species (ROS)-dependent ferroptosis of tumor cells.Conclusion: Such a GSH-starvation therapeutic strategy would cause multi-path programmed cell death and could be a promising strategy for cancer therapy.

Study of Mental Illness in Rat Model of Sodium Azide Induced Oxidative Stress

Aim: Oxidative stress is known as Reactive oxygen species (ROS) accumulation that is caused by reactive ROS and antioxidants imbalance that could be due to decreased antioxidant levels. Oxidative stress is often related to aging, Oxygen metabolism and redox imbalance in cells and tissues. It is a cellular state in which oxidants levels e.g. superoxide (O-2), hydrogen peroxide (H2O2) or nitric oxide (NO) in biological metabolisms exceed the oxidants scavenging capacity of cells. Oxidative stress in brain leads to depression, anxiety, memory impairment and behavioral deficits associated with them. Method: 24 male albino wistar rats were allocated into test and controls groups administered with sodium azide (5 mg/kg bodyweight) (i.p.) and water (p.o.) respectively for 14 days. Behaviors were monitored weekly after 24 hours of sodium azide administration in light/dark box, elevated plus maze, Open field and Morris water maze. Results: Test animals that were administered with sodium azide significantly decreased entries and time spent in illuminated area of light dark box and elevated plus maze while increased latency and fewer square crossed were observed with decreased learning acquisition and memory retention. Conclusion: All the data collected and results analysis determine oxidative stress could cause mood disorders learning disabilities. Sodium azide induced oxidative stress produce behavioral deficits and memory impairment validated it as a neurotoxin.

Proteasomal inhibition attenuates craniofacial malformations in a zebrafish model of Treacher Collins Syndrome

Treacher Collins Syndrome (TCS) is a congenital disease characterized by defects in the craniofacial skeleton and absence of mental alterations. Recently we modelled TCS in zebrafish (Danio rerio) embryos through the microinjection of Morpholino® oligonucleotides blocking the translation of the ortholog of the main causative gene (TCOF1). We showed that Cnbp, a key cytoprotective protein involved in normal rostral head development, was detected in lower levels (without changes in its mRNA expression) in TCS-like embryos. As previous reports suggested that Cnbp is degraded through the proteasomal pathway, we tested whether proteasome inhibitors (MG132 and Bortezomib (Velcade®, Millennium laboratories)) were able to ameliorate cranial skeleton malformations in TCS. Here we show that treatment with both proteasome inhibitors produced a robust craniofacial cartilage phenotype recovery. This recovery seems to be consequence of a decreased degradation of Cnbp in TCS-like embryos. Critical TCS manifestations, such as neuroepithelial cell death and cell redox imbalance were attenuated. Thus, proteasome inhibitors may offer an opportunity for TCS molecular and phenotypic manifestation’s prevention. Although further development of new safe inhibitors compatible with administration during pregnancy is required, our results encourage this therapeutic approach.

Melatonin reduces oxidative stress and promotes drought tolerance in young Coffea arabica L. plants

Water deficit severely compromises the homeostatic balance of plants and suggests a redox imbalance in cells. Melatonin is described as a promoter of stress tolerance in plants that induces the expression of stress-related genes, reduces lipid peroxidation and increases the antioxidant system. To analyze the influence of exogenous melatonin on the promotion of tolerance to water deficit, we applied concentrations of 300 μM and 500 μM to Coffea arabica L. seedlings. Here, we show that the intensity of the short-term responses to melatonin may vary according to the concentration of melatonin. We also report that lower concentrations of melatonin (300 μM) promote an increase in the root system and the protection of the photosynthetic apparatus, allowing greater gas exchange, greater carboxylation efficiency and higher chlorophyll contents. Moreover, melatonin improved the activity of the enzymatic and nonenzymatic antioxidant systems and reduced lipid peroxidation. In addition, we demonstrated that higher concentrations (500 μM) caused negative effects on stress tolerance, thus demonstrating a toxic level. Overall, these results demonstrated that melatonin mediates the signaling for water deficit responses by acting as an inductor of tolerance, most likely enhanced by increased carboxylation efficiency and antioxidant systems. This study provides evidence that exogenous melatonin protects coffee against water deficit.

Nanoparticles-Caused Oxidative Imbalance.

Application of nanomaterials in nearly every single branch of industry results in their accumulation in both abiotic environment and tissues of living organisms. Despite the common use of nanomaterials, we are not able to precisely define their toxicity towards humans and surrounding biota. Although we were able to determine final effects of chronic exposure to nanoparticles which consist of many pathologies such as respiratory diseases, allergies, diseases of cardiovascular system, disorders in embryonic life differentiation and growth disorders, toxic effects on the immune system and cancers. The most predominantly investigated feature of most nanoparticles is their ability to induce oxidative stress on cellular level. Imbalance in redox state of cells can lead to various malfunctions in their internal metabolism, which in turn can lead to mentioned pathologies on the organismal level if the exposure is persistent and spread wide enough. Imbalance in redox state translate into production of reactive oxygen species in amounts impossible to be scavenged in given time. Many reactive oxygen species play crucial role in physiological processes in properly functioning cells. It was proven on numerous occasions that abundance of ROS, aside from oxidative damage, can lead to more subtle adverse effects tied to disturbances in intra- and intercellular signaling pathways. In this chapter we would like to address the nanoparticle-induced redox imbalance in cells and its effects.

Chemoproteomic profiling of targets of lipid-derived electrophiles by bioorthogonal aminooxy probe

Redox imbalance in cells induces lipid peroxidation and generates a class of highly reactive metabolites known as lipid-derived electrophiles (LDEs) that can modify proteins and affects their functions. Identifying targets of LDEs is critical to understand how such modifications are functionally implicated in oxidative-stress associated diseases. Here we report a quantitative chemoproteomic method to globally profile protein targets and sites modified by LDEs. In this strategy, we designed and synthesized an alkyne-functionalized aminooxy probe to react with LDE-modified proteins for imaging and proteomic profiling. Using this probe, we successfully quantified >4000 proteins modified by 4-hydroxy-2-nonenal (HNE) of high confidence in mammalian cell lysate and combined with a tandem-orthogonal proteolysis activity-based protein profiling (TOP-ABPP) strategy, we identified ~400 residue sites targeted by HNE including reactive cysteines in peroxiredoxins, an important family of enzymes with anti-oxidant roles. Our method expands the toolbox to quantitatively profile protein targets of endogenous electrophiles and the enlarged inventory of LDE-modified proteins and sites will contribute to functional elucidation of cellular pathways affected by oxidative stress.

DNA and mitochondrial protective effect of lycopene rich tomato (Solanum lycopersicum L.) peel extract prepared by enzyme assisted extraction against H2O2 induced oxidative damage in L6 myoblasts

Lycopene rich extracts (ETE) were prepared by enzyme assisted extraction of tomato peel. Oxidative damage was induced in L6 myoblasts by exposing cells to H2O2 in presence and absence (control) of ETE. The potential of ETE to protect cells from oxidative damage was investigated in terms of cytotoxicity (MTT assay), Reactive oxygen species (ROS) by 2′, 7′-dichlorofluorescindiacetate (H2DCFDA), DNA damage (ladder assay, 8-oxo-dG), Hoechst 33342 nuclear staining, mitochondrial stability by ATP production and mitochondrial membrane potential by Rhodamine 123 staining. It was observed that treatment of cells with ETE significantly increased cell viability, decreased ROS production, reduced DNA fragmentation, chromatin condensation & 8-oxo-dG, increased ATP levels and mitochondrial membrane potential. Results indicated that ETE could protect cells from H2O2 induced oxidative damage significantly as compared to control. These results depicted the antioxidant potential of tomato peel extract which can counteract the redox imbalance in cells induced by oxidative stress condition.

Mechanisms of physiological adjustment of N2 fixation in Cicer arietinum L. (chickpea) during early stages of water deficit: single or multi-factor controls.

Drought negatively impacts symbiotic nitrogen fixation (SNF) in Cicer arietinumL. (chickpea), thereby limiting yield potential. Understanding how drought affects chickpea nodulation will enable the development of strategies to biotechnologically engineer chickpea varieties with enhanced SNF under drought conditions. By analyzing carbon and nitrogen metabolism, we studied the mechanisms of physiological adjustment of nitrogen fixation in chickpea plants nodulated with Mesorhizobium ciceri during both drought stress and subsequent recovery. The nitrogenase activity, levels of several key carbon (in nodules) and nitrogen (in both nodules and leaves) metabolites and antioxidant compounds, as well as the activity of related nodule enzymes were examined in M.ciceri-inoculated chickpea plants under early drought stress and subsequent recovery. Results indicated that drought reduced nitrogenase activity, and that this was associated with a reduced expression of the nifK gene. Furthermore, drought stress promoted an accumulation of amino acids, mainly asparagine in nodules (but not in leaves), and caused a cell redox imbalance in nodules. An accumulation of organic acids, especially malate, in nodules, which coincided with the decline of nodulated root respiration, was also observed under drought stress. Taken together, our findings indicate that reduced nitrogenase activity occurring at early stages of drought stress involves, at least, the inhibition of respiration, nitrogen accumulation and an imbalance in cell redox status in nodules. The results of this study demonstrate the potential that the genetic engineering-based improvement of SNF efficiency could be applied to reduce the impact of drought on the productivity of chickpea, and perhaps other legume crops.

Mitochondrial ATP-sensitive K + channels as redox signals to liver mitochondria in response to hypertriglyceridemia

We have recently demonstrated that hypertriglyceridemic (HTG) mice present both elevated body metabolic rates and mild mitochondrial uncoupling in the liver owing to stimulated activity of the ATP-sensitive potassium channel (mitoK ATP). Because lipid excess normally leads to cell redox imbalance, we examined the hepatic oxidative status in this model. Cell redox imbalance was evidenced by increased total levels of carbonylated proteins, malondialdehydes, and GSSG/GSH ratios in HTG livers compared to wild type. In addition, the activities of the extramitochondrial enzymes NADPH oxidase and xanthine oxidase were elevated in HTG livers. In contrast, Mn-superoxide dismutase activity and content, a mitochondrial matrix marker, were significantly decreased in HTG livers. Isolated HTG liver mitochondria presented lower rates of H 2O 2 production, which were reversed by mitoK ATP antagonists. In vivo antioxidant treatment with N-acetylcysteine decreased both mitoK ATP activity and metabolic rates in HTG mice. These data indicate that high levels of triglycerides increase reactive oxygen generation by extramitochondrial enzymes that promote mitoK ATP activation. The mild uncoupling mediated by mitoK ATP increases metabolic rates and protects mitochondria against oxidative damage. Therefore, a biological role for mitoK ATP as a redox sensor is shown here for the first time in an in vivo model of systemic and cellular lipid excess.

Nitrogen Fixation Control under Drought Stress. Localized or Systemic?

Legume-Rhizobium nitrogen fixation is dramatically affected under drought and other environmental constraints. However, it has yet to be established as to whether such regulation of nitrogen fixation is only exerted at the whole-plant level (e.g. by a systemic nitrogen feedback mechanism) or can also occur at a local nodule level. To address this question, nodulated pea (Pisum sativum) plants were grown in a split-root system, which allowed for half of the root system to be irrigated at field capacity, while the other half was water deprived, thus provoking changes in the nodule water potential. Nitrogen fixation only declined in the water-deprived, half-root system and this result was correlated with modifications in the activities of key nodule's enzymes such as sucrose synthase and isocitrate dehydrogenase and in nodular malate content. Furthermore, the decline in nodule water potential resulted in a cell redox imbalance. The results also indicate that systemic nitrogen feedback signaling was not operating in these water-stressed plants, since nitrogen fixation activity was maintained at control values in the watered half of the split-root plants. Thus, the use of a partially droughted split-root system provides evidence that nitrogen fixation activity under drought stress is mainly controlled at the local level rather than by a systemic nitrogen signal.

NaoXinQing, an anti-stroke herbal medicine, reduces hydrogen peroxide-induced injury in NG108-15 cells

NaoXinQing (NXQ) is a patented and approved drug of Traditional Chinese Medicine that has been used for years for the treatment of syndrome of apoplexy, but the underlying mechanism remains unclear. The present study is designed to investigate the effects of NXQ on hydrogen peroxide (H 2O 2)-induced cell damage in NG108-15 cells. Exposure to H 2O 2 induces apoptosis-like cell injury. Preincubation of cells with NXQ alleviated H 2O 2-induced cell injury and apoptosis. This herb medicine also improves redox imbalance in cells under the exposure of H 2O 2 as indicated by the attenuation in the reduction of activities of intracellular endogenous antioxidants, glutathione and glutathione peroxidase as well as catalase, and by the decrease in the leak of lactate dehydrogenase and the accumulation of malondialdehyde. These results indicate that NXQ significantly protects NG108-15 cells against H 2O 2 challenge by improving redox imbalance and inhibiting apoptosis, which might represent mechanisms underlying its potential usage in the prevention and treatment of syndrome of apoplexy.