Effects of Nitrosyl Iron Complexes with Thiol, Phosphate, and Thiosulfate Ligands on Hemoglobin.

Nitrosyl iron complexes are remarkably multifactorial pharmacological agents. These compounds have been proven to be particularly effective in treating cardiovascular and oncological diseases. We evaluated and compared the antioxidant activity of tetranitrosyl iron complexes (TNICs) with thiosulfate ligands and dinitrosyl iron complexes (DNICs) with glutathione (DNIC-GS) or phosphate (DNIC-PO4-) ligands in hemoglobin-containing systems. The studied effects included the production of free radical intermediates during hemoglobin (Hb) oxidation by tert-butyl hydroperoxide, oxidative modification of Hb, and antioxidant properties of nitrosyl iron complexes. Measuring luminol chemiluminescence revealed that the antioxidant effect of TNICs was higher compared to DNIC-PO4-. DNIC-GS either did not exhibit antioxidant activity or exerted prooxidant effects at certain concentrations, which might have resulted from thiyl radical formation. TNICs and DNIC-PO4- efficiently protected the Hb heme group from decomposition by organic hydroperoxides. DNIC-GS did not exert any protective effects on the heme group; however, it abolished oxoferrylHb generation. TNICs inhibited the formation of Hb multimeric forms more efficiently than DNICs. Thus, TNICs had more pronounced antioxidant activity than DNICs in Hb-containing systems.

Biphasic dose-response and effects of near-infrared photobiomodulation on erythrocytes susceptibility to oxidative stress in vitro.

The effect of simultaneous application of tert-butyl hydroperoxide (tBHP) and polychromatic near-infrared (NIR) radiation on bovine blood was examined to determine whether NIR light decreases the susceptibility of red blood cells (RBCs) to oxidative stress. The study assessed various exposure methods, wavelength ranges, and optical filtering types. Continuous NIR exposure revealed a biphasic response in cell-free hemoglobin changes, with antioxidative effects observed at low fluences and detrimental effects at higher fluences. Optimal exposure duration was identified between 60 s and 15 min. Protective effects were also tested across wavelengths in the range of 750-1100 nm, with all of them reducing hemolysis, notably at 750 nm, 875 nm, and 900 nm. Comparing broadband NIR and far-red light (750 nm) showed no significant difference in hemolysis reduction. Pulse-dosed NIR irradiation allowed safe increases in radiation dose, effectively limiting hemolysis at higher doses where continuous exposure was harmful. These findings highlight NIR photobiomodulation's potential in protecting RBCs from oxidative stress and will be helpful in the effective design of novel medical therapeutic devices.

Antioxidant and anti-inflammatory function of Eupatorium adenophora Spreng leaves (EASL) on human intestinal Caco-2 cells treated with tert-butyl hydroperoxide.

Chronic non-communicable diseases (CNCDs) pose a significant public health challenge. Addressing this issue, there has been a notable breakthrough in the prevention and mitigation of NCDs through the use of antioxidants and anti-inflammatory agents. In this study, we aim to explore the effectiveness of Eupatorium adenophora Spreng leaves (EASL) as an antioxidant and anti-inflammatory agent, and its potential applications. To construct a cellular model of oxidative damage and inflammation, Caco-2 cells were treated with tert-butyl hydroperoxide (t-BHP). The biocompatibility of EASL-AE with Caco-2 cells was assessed using the MTT assay, while compatibility was further verified by measuring LDH release and the protective effect against oxidative damage was also assessed using the MTT assay. Additionally, we measured intracellular oxidative stress indicators such as ROS and 8-OHdG, as well as inflammatory pathway signalling protein NFκB and inflammatory factors TNF-α and IL-1ß using ELISA, to evaluate the antioxidant and anti-inflammatory capacity of EASL-AE. The scavenging capacity of EASL-AE against free radicals was determined through the DPPH Assay and ABTS Assay. Furthermore, we measured the total phenolic, total flavonoid, and total polysaccharide contents using common chemical methods. The chemical composition of EASL-AE was analyzed using the LC-MS/MS technique. Our findings demonstrate that EASL-AE is biocompatible with Caco-2 cells and non-toxic at experimental levels. Moreover, EASL-AE exhibits a significant protective effect on Caco-2 cells subjected to oxidative damage. The antioxidant effect of EASL-AE involves the scavenging of intracellular ROS, while its anti-inflammatory effect is achieved by down-regulation of the NFκB pathway. Which in turn reduces the release of inflammatory factors TNF-α and IL-1ß. Through LC-MS/MS analysis, we identified 222 compounds in EASL-AE, among which gentianic acid, procaine and L-tyrosine were the compounds with high antioxidant capacity and may be the effective constituent for EASL-AE with antioxidant activity. These results suggest that EASL-AE is a natural and high-quality antioxidant and anti-inflammatory biomaterial that warrants further investigation. It holds great potential for applications in healthcare and other related fields.

Crassostrea gigas peptide PEP-1 prevents tert-butyl hydroperoxide (t-BHP) induced oxidative stress in HepG2 cells.

Exposure to tert-butyl hydroperoxide (t-BHP) leads to cytotoxicity and oxidative stress in various organs and cell types. The bioactive peptides extracted from Oysters exhibit marked antioxidant activity. The impacts of Crassostrea gigas peptides on t-BHP-triggered oxidative stress remain largely unknown. The protective and antioxidant activity of a C.gigas peptide, PEP-1, on t-BHP-treated HepG2 cells, was investigated. PEP-1, this peptide is arginine kinase in oysters. This enzyme functions as a catalyst for the chemical reaction and serves as a phosphate transferase. Since it was the most expressed protein in the adductor muscle of oysters. Our determination showed the lowest level of a toxic concentration of t-BHP (200 µM) and the resting concentration of PEP-1 (0-1000 ng/ml). PEP-1 exerted a protective effect against t-BHP-induced apoptosis by modifying the expression of pro-and anti-apoptotic proteins. PEP-1 administration reduced nitric oxide and ROS levels while restoring levels of antioxidant proteins in t-BHP-induced cells. PEP-1 exhibited the capacity to enhance the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, the C. gigas peptide PEP-1 has demonstrated its ability to protect HepG2 cells against oxidative stress induced by t-BHP.

Deferoxamine protects cochlear hair cells and hair cell-like HEI-OC1 cells against tert-butyl hydroperoxide-induced ototoxicity.

Oxidative stress is the common mechanism of sensorineural hearing loss (SNHL) caused by many factors, such as noise, drugs and ageing. Here, we used tert-butyl hydroperoxide (t-BHP) to cause oxidative stress damage in HEI-OC1 cells and in an in vitro cochlear explant model. We observed lipid peroxidation, iron accumulation, mitochondrial shrinkage and vanishing of mitochondrial cristae, which caused hair cell ferroptosis, after t-BHP exposure. Moreover, the number of TUNEL-positive cells in cochlear explants and HEI-OC1 cells increased significantly, suggesting that t-BHP caused the apoptosis of hair cells. Administration of deferoxamine (DFOM) significantly attenuated t-BHP-induced hair cell loss and disordered hair cell arrangement in cochlear explants as well as HEI-OC1 cell death, including via apoptosis and ferroptosis. Mechanistically, we found that DFOM treatment reduced t-BHP-induced lipid peroxidation, iron accumulation and mitochondrial pathological changes in hair cells, consequently mitigating apoptosis and ferroptosis. Moreover, DFOM treatment alleviated GSH depletion caused by t-BHP and activated the Nrf2 signalling pathway to exert a protective effect. Furthermore, we confirmed that the protective effect of DFOM mainly depended on its ability to chelate iron by constructing Fth1 knockout (KO), TfR1 KO and Nrf2 KO HEI-OC1 cell lines using CRISPR/Cas9 technology and a Flag-Fth1 (overexpression) HEI-OC1 cell line using the FlpIn™ System. Our findings suggest that DFOM is a potential drug for SNHL treatment due to its ability to inhibit apoptosis and ferroptosis by chelating iron and scavenging reactive oxygen species (ROS).

The Tumor Suppressor SOCS1 Diminishes Tolerance to Oxidative Stress in Hepatocellular Carcinoma.

SOCS1 is a tumor suppressor in hepatocellular carcinoma (HCC). Recently, we showed that a loss of SOCS1 in hepatocytes promotes NRF2 activation. Here, we investigated how SOCS1 expression in HCC cells affected oxidative stress response and modulated the cellular proteome. Murine Hepa1-6 cells expressing SOCS1 (Hepa-SOCS1) or control vector (Hepa-Vector) were treated with cisplatin or tert-butyl hydroperoxide (t-BHP). The induction of NRF2 and its target genes, oxidative stress, lipid peroxidation, cell survival and cellular proteome profiles were evaluated. NRF2 induction was significantly reduced in Hepa-SOCS1 cells. The gene and protein expression of NRF2 targets were differentially induced in Hepa-Vector cells but markedly suppressed in Hepa-SOCS1 cells. Hepa-SOCS1 cells displayed an increased induction of reactive oxygen species but reduced lipid peroxidation. Nonetheless, Hepa-SOCS1 cells treated with cisplatin or t-BHP showed reduced survival. GCLC, poorly induced in Hepa-SOCS1 cells, showed a strong positive correlation with NFE2L2 and an inverse correlation with SOCS1 in the TCGA-LIHC transcriptomic data. A proteomic analysis of Hepa-Vector and Hepa-SOCS1 cells revealed that SOCS1 differentially modulated many proteins involved in diverse molecular pathways, including mitochondrial ROS generation and ROS detoxification, through peroxiredoxin and thioredoxin systems. Our findings indicate that maintaining sensitivity to oxidative stress is an important tumor suppression mechanism of SOCS1 in HCC.

Propolis Protects GC-1spg Spermatogonial Cells against Tert-Butyl Hydroperoxide-Induced Oxidative Damage.

Propolis is a natural resin produced by honeybees with plenty of pharmacologic properties, including antioxidant activity. Oxidative stress disrupts germ cell development and sperm function, with demonstrated harmful effects on male reproduction. Several natural antioxidants have been shown to reduce oxidative damage and increase sperm fertility potential; however, little is known about the effects of propolis. This work evaluated the role of propolis in protecting spermatogonial cells from oxidative damage. Propolis' phytochemical composition and antioxidant potential were determined, and mouse GC-1spg spermatogonial cells were treated with 0.1-500 µg/mL propolis (12-48 h) in the presence or absence of an oxidant stimulus (tert-butyl hydroperoxide, TBHP, 0.005-3.6 µg/mL, 12 h). Cytotoxicity was assessed by MTT assays and proliferation by Ki-67 immunocytochemistry. Apoptosis, reactive oxygen species (ROS), and antioxidant defenses were evaluated colorimetrically. Propolis presented high phenolic and flavonoid content and moderate antioxidant activity, increasing the viability of GC-1spg cells and counteracting TBHP's effects on viability and proliferation. Additionally, propolis reduced ROS levels in GC-1spg, regardless of the presence of TBHP. Propolis decreased caspase-3 and increased glutathione peroxidase activity in TBHP-treated GC-1spg cells. The present study shows the protective action of propolis against oxidative damage in spermatogonia, opening the possibility of exploiting its benefits to male fertility.

Influence of Microbiota-Related Metabolites Associated with Inflammation and Sepsis on the Peroxidase Activity of Cyclooxygenase in Healthy Human Monocytes and Acute Monocytic Leukemia Cells.

The human microbiota produces metabolites that can enter the bloodstream and exert systemic effects on various functions in both healthy and pathological states. We have studied the participation of microbiota-related metabolites in bacterial infection by examining their influence on the activity of cyclooxygenase (COX) as a key enzyme of inflammation. The influence of aromatic microbial metabolites, derivatives of phenylalanine (phenylpropionic acid, PPA), tyrosine (4-hydroxyphenyllactic acid, HPLA), and tryptophan (indolacetic acids, IAA), the concentrations of which in the blood change notably during sepsis, was evaluated. Also, the effect of itaconic acid (ITA) was studied, which is formed in macrophages under the action of bacterial lipopolysaccharides (LPS) and appears in the blood in the early stages of infection. Metabiotic acetyl phosphate (AcP) as a strong acetylating agent was also tested. The activity of COX was measured via the TMPD oxidation colorimetric assay using the commercial pure enzyme, cultured healthy monocytes, and the human acute monocytic leukemia cell line THP-1. All metabolites in the concentration range of 100-500 µM lowered the activity of COX. The most pronounced inhibition was observed on the commercial pure enzyme, reaching up to 40% in the presence of AcP and 20-30% in the presence of the other metabolites. On cell lysates, the effect of metabolites was preserved, although it significantly decreased, probably due to their interaction with other targets subject to redox-dependent and acetylation processes. The possible contribution of the redox-dependent action of microbial metabolites was confirmed by assessing the activity of the enzyme in the presence of thiol reagents and in model conditions, when the COX-formed peroxy intermediate was replaced with tert-butyl hydroperoxide (TBH). The data show the involvement of the microbial metabolites in the regulation of COX activity, probably due to their influence on the peroxidase activity of the enzyme.

Cytoprotective Effect of Bambusae caulis in Liquamen by Blocking Oxidative Stress in Hepatocytes.

Bambusae caulis in Liquamen (BCL), which is extracted from heat-treated fresh bamboo stems, is a traditional herbal medicine widely used in Eastern countries. Recently, it has been reported to have anti-inflammatory and whitening effects. However, the protective effect of BCL on hepatocytes has not yet been elucidated. The present study aimed to determine whether BCL prevents oxidative stress induced by tert-butyl hydroperoxide (t-BHP) and exerts cytoprotective effects on hepatocytes. High-performance liquid chromatography and liquid chromatography with tandem mass spectroscopy were performed to analyze the type of polyphenols present in BCL. The activities of antioxidant enzymes and hepatocyte viability were assessed. The benzoic acid content was the highest among polyphenols present in BCL. Benzoic acid acts as a scavenger of free radicals, including reactive oxygen species. BCL increased the expression of antioxidant enzymes (glutamate-cysteine ligase and NADPH quinone dehydrogenase (1)) by activating nuclear factor erythroid 2-related factor 2 and reduced tBHP-induced cell death by inhibiting oxidative stress. BCL inhibited tBHP-induced phosphorylation of p38 and c-Jun N-terminal kinase but not that of extracellular signal-regulated kinase. In conclusion, BCL is a promising therapeutic candidate for treating oxidative-stress-induced hepatocyte damage.

Oxido-Reduction Potential as a Method to Determine Oxidative Stress in Semen Samples.

There are different estimates for the incidence of infertility. Its occurrence may vary from area to area, but on average, it affects 15% of couples and 10-12% of men worldwide. Many aspects of infertility can be linked to reactive oxygen species (ROS) and the process of oxidative stress (OS). The association between poor semen quality and OS is well known. Unfortunately, there is no accepted protocol for the diagnosis and treatment of OS in andrology. Oxido-reduction potential (ORP) measurement is a new method for determining the ratio between oxidant and antioxidant molecules. Currently, ORP measurement is one of the fastest and most user-friendly methods of andrological OS determination and our goals were to confirm published correlations between ORP values and sperm parameters, examine how sperm concentration influences these results, and investigate whether intracellular ROS formations are also manifested in the ORP values or not after artificial ROS induction. Intracellular ROS formations were induced by menadione (superoxide anion inducer), hydrogen peroxide, and tert-butyl hydroperoxide (lipid peroxidation inducer) treatments; sperm parameters like motility and viability were determined with an SCA Scope system, and ORP changes were recorded by the Mioxsys system. Significant correlations were noticed among the ORP, spermatozoa concentration, motility, progressive motility, and viability. Nevertheless, only the ORP value after normalization with the sperm count correlated with these parameters. Due to normalization, very low and very high sperm concentrations can give misleading results. The means of the non-normalized ORP values were almost the same. All of the applied treatments resulted in decreases in the viability, motility, and progressive motility, and interestingly, altered ORP levels were detected. In addition, it was determined that seminal plasma had a significant protective effect on spermatozoa. The elimination of seminal plasma caused higher sensitivity of spermatozoa against used OS inducers, and higher ORP levels and decreased viabilities and motilities were measured. The ORP level could be a good indicator of male OS; however, in cases of low and high sperm counts, its result can be misleading. Overall, the conclusion can be drawn that ORP determination is a suitable method for detecting intracellular ROS accumulation, but it has limitations that still need to be clarified.

Proteome changes in a human retinal pigment epithelial cell line during oxidative stress and following antioxidant treatment.

Age related macular degeneration (AMD) is the most common cause of blindness in the elderly. Oxidative stress contributes to retinal pigment epithelium (RPE) dysfunction and cell death thereby leading to AMD. Using improved RPE cell model systems, such as human telomerase transcriptase-overexpressing (hTERT) RPE cells (hTERT-RPE), pathophysiological changes in RPE during oxidative stress can be better understood. Using this model system, we identified changes in the expression of proteins involved in the cellular antioxidant responses after induction of oxidative stress. Some antioxidants such as vitamin E (tocopherols and tocotrienols) are powerful antioxidants that can reduce oxidative damage in cells. Alpha-tocopherol (α-Toc or αT) and gamma-tocopherol (γ-Toc or γT) are well-studied tocopherols, but signaling mechanisms underlying their respective cytoprotective properties may be distinct. Here, we determined what effect oxidative stress, induced by extracellularly applied tBHP in the presence and absence of αT and/or γT, has on the expression of antioxidant proteins and related signaling networks. Using proteomics approaches, we identified differential protein expression in cellular antioxidant response pathways during oxidative stress and after tocopherol treatment. We identified three groups of proteins based on biochemical function: glutathione metabolism/transfer, peroxidases and redox-sensitive proteins involved in cytoprotective signaling. We found that oxidative stress and tocopherol treatment resulted in unique changes in these three groups of antioxidant proteins indicate that αT and γT independently and by themselves can induce the expression of antioxidant proteins in RPE cells. These results provide novel rationales for potential therapeutic strategies to protect RPE cells from oxidative stress.

Specific Features of Mitochondrial Dysfunction under Conditions of Ferroptosis Induced by t-Butylhydroperoxide and Iron: Protective Role of the Inhibitors of Lipid Peroxidation and Mitochondrial Permeability Transition Pore Opening.

Recent studies have indicated the critical importance of mitochondria in the induction and progression of ferroptosis. There is evidence indicating that tert-butyl hydroperoxide (TBH), a lipid-soluble organic peroxide, is capable of inducing ferroptosis-type cell death. We investigated the effect of TBH on the induction of nonspecific membrane permeability measured by mitochondrial swelling and on oxidative phosphorylation and NADH oxidation assessed by NADH fluo rescence. TBH and iron, as well as their combinations, induced, with a respective decrease in the lag phase, the swelling of mitochondria, inhibited oxidative phosphorylation and stimulated NADH oxidation. The lipid radical scavenger butylhydroxytoluene (BHT), the inhibitor of mitochondrial phospholipase iPLA2γ bromoenol lactone (BEL), and the inhibitor of the mitochondrial permeability transition pore (MPTP) opening cyclosporine A (CsA) were equally effective in protecting these mitochondrial functions. The radical-trapping antioxidant ferrostatin-1, a known indicator of ferroptotic alteration, restricted the swelling but was less effective than BHT. ADP and oligomycin significantly decelerated iron- and TBH-induced swelling, confirming the involvement of MPTP opening in mitochondrial dysfunction. Thus, our data showed the participation of phospholipase activation, lipid peroxidation, and the MPTP opening in the mitochondria-dependent ferroptosis. Presumably, their involvement took place at different stages of membrane damage initiated by ferroptotic stimuli.

Protective effect of Buddha's Temple extract against tert-butyl hydroperoxide stimulation-induced oxidative stress in DF-1 cells.

OBJECTIVE: This study aimed to determine the protective efficacy of Buddha's Temple (BT) extract against tert-butyl hydroperoxide (t-BHP)-induced oxidative stress in Gallus gallus chicken embryo fibroblast cell line (DF-1) and its effects on the cell lipid metabolism. METHODS: In this experimental study, Gallus gallus DF-1 fibroblast cells were pretreated with BT 10-7 for 24 hours, followed by their six-hour exposure to t-BHP (100 µM). Water-soluble tetrazolium salt-8 (WST-8) assays were performed, and the growth curve was computed. The intracellular gene expression changes caused by BT extract were confirmed through quantitative polymerase chain reaction (qPCR). Flow cytometry, oil red O staining experiment, and thin-layer chromatography were performed for the detection of intracellular metabolic mechanism changes. RESULTS: The WST-8 assay results showed that the BT pretreatment of Gallus gallus DF-1 fibroblast cell increased their cell survival rate by 1.08%±0.04%, decreased the reactive oxygen species (ROS) level by 0.93%±0.12% even after exposure to oxidants, and stabilized mitochondrial activity by 1.37%±0.36%. In addition, qPCR results confirmed that the gene expression levels of tumor necrosis factor α (TNFα), TIR domain-containing adapter inducing IFN-beta (TICAM1), and glucose-regulated protein 78 (GRP78) were regulated, which contributed to cell stabilization. Thin-layer chromatography and oil red O analyses showed a clear decrease in the contents of lipid metabolites such as triacylglycerol and free fatty acids. CONCLUSION: In this study, we confirmed that the examined BT extract exerted selective protective effects on Gallus gallus DF-1 fibroblast cells against cell damage caused by t-BHP, which is a strong oxidative inducer. Furthermore, we established that this extract significantly reduced the intracellular ROS accumulation due to oxidative stress, which contributes to an increase in poultry production and higher incomes.

Potential of Tamarind Shell Extract against Oxidative Stress In Vivo and In Vitro.

Tamarind shell is rich in flavonoids and exhibits good biological activities. In this study, we aimed to analyze the chemical composition of tamarind shell extract (TSE), and to investigate antioxidant capacity of TSE in vitro and in vivo. The tamarind shells were extracted with 95% ethanol refluxing extraction, and chemical constituents were determined by ultra-performance chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). The free radical scavenging activity of TSE in vitro was evaluated using the oxygen radical absorbance capacity (ORAC) method. The antioxidative effects of TSE were further assessed in 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH)-stimulated ADTC5 cells and tert-butyl hydroperoxide (t-BHP)-exposed zebrafish. A total of eight flavonoids were detected in TSE, including (+)-catechin, taxifolin, myricetin, eriodictyol, luteolin, morin, apigenin, and naringenin, with the contents of 5.287, 8.419, 4.042, 6.583, 3.421, 4.651, 0.2027, and 0.6234 mg/g, respectively. The ORAC assay revealed TSE and these flavonoids had strong free radical scavenging activity in vitro. In addition, TSE significantly decreased the ROS and MDA levels but restored the SOD activity in AAPH-treated ATDC5 cells and t-BHP-exposed zebrafish. The flavonoids also showed excellent antioxidative activities against oxidative damage in ATDC5 cells and zebrafish. Overall, the study suggests the free radical scavenging capacity and antioxidant potential of TSE and its primary flavonoids in vitro and in vivo and will provide a theoretical basis for the development and utilization of tamarind shell.

LPS-Activated Microglial Cell-Derived Conditioned Medium Protects HT22 Neuronal Cells against Glutamate-Induced Ferroptosis.

Neuron-glia interactions are essential for the central nervous system's homeostasis. Microglial cells are one of the key support cells in the brain that respond to disruptions in such homeostasis. Although their participation in neuroinflammation is well known, studies investigating their role in ferroptosis, an iron-dependent form of nonapoptotic cell death, are lacking. To address this issue, we explored whether microglial (BV-2 cells) activation products can intensify, mitigate or block oxidative and/or ferroptotic damage in neuronal cells (HT22 cell line). Cultured BV-2 microglial cells were stimulated with 5-100 ng/mL lipopolysaccharide (LPS) for 24 h and, after confirmation of microglial activation, their culture medium (conditioned media; CM) was transferred to neuronal cells, which was subsequently (6 h later) exposed to glutamate or tert-butyl hydroperoxide (t-BuOOH). As a major finding, HT22 cells pretreated for 6 h with CM exhibited a significant ferroptosis-resistant phenotype characterized by decreased sensitivity to glutamate (15 mM)-induced cytotoxicity. However, no significant protective effects of LPS-activated microglial cell-derived CM were observed in t-BuOOH (30 µM)-challenged cells. In summary, activated microglia-derived molecules may protect neuronal cells against ferroptosis. The phenomenon observed in this work highlights the beneficial relationship between microglia and neurons, highlighting new possibilities for the control of ferroptosis.

Fate of micronuclei and micronucleated cells after treatment of HeLa cells with different genotoxic agents.

Although micronuclei are well-known biomarkers of genotoxic damage, the biological consequences of micronucleus induction are only poorly understood. To further elucidate these consequences, HeLa cells stably expressing histone 2B coupled with green fluorescent protein were used for long-term live cell imaging to investigate the fate of micronuclei and micronucleated cells after treatment of cells with various genotoxic agents (doxorubicin (20, 30 and nM), tert-butyl hydroperoxide (tBHP, 50, 100 and 150 µM), radiation (0.5, 1 and 2 Gy), methyl methanesulfonate (MMS, 20, 25 and 30 µg/ml) and vinblastine (1, 2 and 3 nM)). Most micronuclei persist for multiple cell cycles or reincorporate while micronucleated cells were more prone to cell death, senescence and fatal mitotic errors compared to non-micronucleated cells, which is consistent with previous studies using etoposide. No clear substance-related effects on the fate of micronuclei and micronucleated cells were observed. To further investigate the fate of micronuclei, extrusion of micronuclei was studied with treatments reported as inducing the extrusion of micronuclei. Since extrusion was not observed in HeLa cells, the relevance of extrusion of micronuclei remains unclear. In addition, degradation of micronuclei was analysed via immunostaining of γH2AX, which demonstrated a high level of DNA damage in micronuclei compared to the main nuclei. Furthermore, transduction with two reporter genes (LC3B-dsRed and LaminB1-dsRed) was conducted followed by long-term live cell imaging. While autophagy marker LC3B was not associated with micronuclei, Lamin B1 was found in approximately 50% of all micronuclei. While degradation of micronuclei was not observed to be a frequent fate of micronuclei, the results show impaired stability of DNA and micronuclear envelope indicating rupture of micronuclei as a pre-step to chromothripsis.

Fluorometric Protocol for Estimating Peroxiredoxin Activity in Biological Tissues.

This protocol describes a detailed fluorometric method for measuring peroxiredoxin (Prx) enzyme activity in vitro. Peroxide dissociation is the rate-limiting step in the Prx-controlled enzymatic reaction. To prevent interference by the catalase enzyme, we developed a peroxiredoxin assay that measures Prx activity using the substrate tert-Butyl hydroperoxide (t-BOOH). Prx enzyme activity is measured by incubating the enzymatic substrates 1,4-dithio-DL-threitol (DTT) and t-BOOH in a suitable buffer at 37 °C for 10 min in the presence of the desired volume of Prx enzyme. Next, the reagent N-(9-Acridinyl)maleimide (NAM) is used to stop the enzymatic reaction and form a fluorescent end product. Finally, Prx activity is measured by thiol fluorometry using a Box-Behnken design to optimize reaction conditions. This novel protocol was validated by evaluating Prx activity in matched samples against a reference assay. The correlation coefficient between our protocol and the reference assay was 0.9933, demonstrating its precision compared with existing methods. The NAM-Prx protocol instead uses t-BOOH as a substrate to measure Prx activity. Because catalase does not participate in the dissociation of t-BOOH, this approach does not require sodium azide. Furthermore, the method eliminates the need for concentrated acids to terminate the Prx enzymatic reaction since the NAM reagent can inhibit the enzymatic reaction regulated by the Prx enzyme.

Antioxidant Efficacy of Esculetin against Tert-Butyl Hydroperoxide-Induced Oxidative Stress in HEK293 Cells.

Esculetin is an antioxidant and anti-inflammatory compound derived from coumarin. Oxidative stress can cause overproduction of reactive oxygen species (ROS), which can lead to the development of chronic kidney failure. In this study, human embryonic kidney 293 (HEK293) cells were treated with tert-butyl hydroperoxide (t-BHP) to determine the antioxidant effects of esculetin. HEK293 cells were treated with t-BHP to validate changes in cell viability, ROS production, and apoptosis, and then treated with esculetin to evaluate the changes. Changes in mRNA and protein levels were analyzed using a proteome kit, PCR, and Western blotting. Esculetin improved HEK293 cell viability and reduced apoptosis caused by t-BHP-induced oxidative stress. At the mRNA and protein levels, esculetin decreased pro-apoptotic factor expression as well as increased anti-apoptotic factor expression. The antioxidant efficacy of esculetin was validated when it inhibited the apoptosis caused by t-BHP-induced oxidative stress in HEK293 cells.

Orientin downregulating oxidative stress-mediated endoplasmic reticulum stress and mitochondrial dysfunction through AMPK/SIRT1 pathway in rat nucleus pulposus cells in vitro and attenuated intervertebral disc degeneration in vivo.

Although considered as a major contributor to low back pain (LBP), intervertebral disc degeneration (IVDD) has poor medical and surgical treatments. Various studies have revealed that endoplasmic reticulum (ER) stress and extracellular matrix (ECM) degeneration play a vital role in initiating and developing the progression of IVDD. Moreover, restoration of SIRT1/AMPK was confirmed to prevent IVDD and damage via maintaining ER and extracellular homeostasis. In addition, orientin (Ori) has been shown to upregulate SIRT1. However, the effect of Ori in nucleus pulposus cells (NPCs) is not determined. Hence, in this study we aim to explore the function of Ori in IVDD pathological model. The results demonstrate that Ori treatment in vitro increased SIRT1/AMPK in NPCs, maintained ECM and ER balance and decreased oxidative stress (OS) response. Ori rescued the disordered homeostasis stimulated by tert-butyl hydroperoxide (TBHP), and its function can be inhibited by thapsigargin (TG). Compound C and EX-527, inhibitors of AMPK and SIRT1 counteracted the Ori-mediated ER stress elimination. These results confirm that Ori exerts its effects by upregulating AMPK and SIRT1. Puncture-stimulated IVDD rats were used to show that Ori attenuates the pathological development in vivo. In all, we partly unveil the underlying mechanisms of Ori in IVDD.

Novel Copper(II) Complexes with Dipinodiazafluorene Ligands: Synthesis, Structure, Magnetic and Catalytic Properties.

The reactions of CuX2 (X = Cl, Br) with dipinodiazafluorenes yielded four new complexes [CuX2L1]2 (X = Cl (1), Br (2), L1 = (1R,3R,8R,10R)-2,2,9,9-Tetramethyl-3,4,7,8,9,10-hexahydro-1H-1,3:8,10-dimethanocyclopenta [1,2-b:5,4-b']diquinolin-12(2H)-one) and [(CuX2)2L2]n (X = Cl (3), Br (4), L2 = (1R,3R,8R,10R,1'R,3'R,8'R,10'R)-2,2,2',2',9,9,9',9'-Octamethyl-1,1',2,2',3,3',4,4',7,7',8,8',9,9',10,10'-hexadecahydro-1,3:1',3':8,10:8',10'-tetramethano-12,12'-bi(cyclopenta [1,2-b:5,4-b']diquinolinylidene). The complexes were characterized by IR and EPR spectroscopy, HR-ESI-MS and elemental analysis. The crystal structures of compounds 1, 2 and 4 were determined by X-ray diffraction (XRD) analysis. Complexes 1-2 have a monomeric structure, while complex 4 has a polymeric structure due to additional coordinating N,N sites in L2. All complexes contain a binuclear fragment {Cu2(µ-X)2×2} (X = Cl, Br) in their structures. Each copper atom has a distorted square-pyramidal coordination environment formed by two nitrogen atoms and three halogen atoms. The Cu-Nax distance is elongated compared to Cu-Neq. The EPR spectra of compounds 1-4 in CH3CN confirm their paramagnetic nature due to the d9 electronic configuration of the copper(II) ion. The magnetic properties of all compounds were studied by the method of static magnetic susceptibility. For complexes 1 and 2, the effective magnetic moments are µeff ≈ 1.87 and 1.83 µB (per each Cu2+ ion), respectively, in the temperature range 50-300 K, which are close to the theoretical spin value (1.73 µB). Ferromagnetic exchange interactions between Cu(II) ions inside {Cu2(µ-X)2X2} (X = Cl, Br) dimers (J/kB ≈ 25 and 31 K for 1 and 2, respectively) or between dimers (θ' ≈ 0.30 and 0.47 K for 1 and 2, respectively) were found at low temperatures. For compounds 3 and 4, the magnetic susceptibility is well described by the Curie-Weiss law in the temperature range 1.77-300 K with µeff ≈ 1.72 and 1.70 µB for 3 and 4, respectively, and weak antiferromagnetic interactions (θ ≈ -0.4 K for 3 and -0.65 K for 4). Complexes 1-4 exhibit high catalytic activity in the oxidation of alkanes and alcohols with peroxides. The maximum yield of cyclohexane oxidation products reached 50% (complex 3). Based on the data on the study of regio- and bond-selectivity, it was concluded that hydroxyl radicals play a decisive role in the oxidation reaction. The initial products in reactions with alkanes are alkyl hydroperoxides.