A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

MRP1-Dependent Extracellular Release of Glutathione Induces Cardiomyocyte Ferroptosis After Ischemia-Reperfusion.

BACKGROUND: The membrane components of cardiomyocytes are rich in polyunsaturated fatty acids, which are easily oxidized. Thus, an efficient glutathione-based lipid redox system is essential for maintaining cellular functions. However, the relationship between disruption of the redox system during ischemia-reperfusion (IR), oxidized lipid production, and consequent cell death (ferroptosis) remains unclear. We investigated the mechanisms underlying the disruption of the glutathione-mediated reduction system related to ferroptosis during IR and developed intervention strategies to suppress ferroptosis. METHODS: In vivo fluctuations of both intra- and extracellular metabolite levels during IR were explored via microdialysis and tissue metabolome analysis. Oxidized phosphatidylcholines were assessed using liquid chromatography high-resolution mass spectrometry. The areas at risk following IR were assessed using triphenyl-tetrazolium chloride/Evans blue stain. RESULTS: Metabolomic analysis combined with microdialysis revealed a significant release of glutathione from the ischemic region into extracellular spaces during ischemia and after reperfusion. The release of glutathione into extracellular spaces and a concomitant decrease in intracellular glutathione concentrations were also observed during anoxia-reperfusion in an in vitro cardiomyocyte model. This extracellular glutathione release was prevented by chemical inhibition or genetic suppression of glutathione transporters, mainly MRP1 (multidrug resistance protein 1). Treatment with MRP1 inhibitor reduced the intracellular reactive oxygen species levels and lipid peroxidation, thereby inhibiting cell death. Subsequent in vivo evaluation of endogenously oxidized phospholipids following IR demonstrated the involvement of ferroptosis, as levels of multiple oxidized phosphatidylcholines were significantly elevated in the ischemic region 12 hours after reperfusion. Inhibition of the MRP1 transporter also alleviated intracellular glutathione depletion in vivo and significantly reduced the generation of oxidized phosphatidylcholines. Administration of MRP1 inhibitors significantly attenuated infarct size after IR injury. CONCLUSIONS: Glutathione was released continuously during IR, primarily in an MRP1-dependent manner, and induced ferroptosis. Suppression of glutathione release attenuated ferroptosis and reduced myocardial infarct size following IR.

5-exo-Selective asymmetric bromolactonization of stilbenecarboxylic acids catalyzed by phenol-bearing chiral thiourea.

We developed a novel thiourea Lewis-base catalyst with phenol moieties for the enantioselective 5-exo-bromolactonization of stilbenecarboxylic acids to afford chiral 3-substituted phthalides. The phenol moieties are crucial for the enantio- and regio-selectivity.

Ethoxyquin, a Lipid Peroxidation Inhibitor, Has Protective Effects against White Matter Lesions in a Mouse Model of Chronic Cerebral Hypoperfusion.

White matter lesions induced by chronic cerebral hypoperfusion can cause vascular dementia; however, no appropriate treatments are currently available for these diseases. In this study, we investigated lipid peroxidation, which has recently been pointed out to be associated with cerebrovascular disease and vascular dementia, as a therapeutic target for chronic cerebral hypoperfusion. We used ethoxyquin, a lipid-soluble antioxidant, in a neuronal cell line and mouse model of the disease. The cytoprotective effect of ethoxyquin on glutamate-stimulated HT-22 cells, a mouse hippocampal cell line, was comparable to that of a ferroptosis inhibitor. In addition, the administration of ethoxyquin to bilateral common carotid artery stenosis model mice suppressed white matter lesions, blood-brain barrier disruption, and glial cell activation. Taken together, we propose that the inhibition of lipid peroxidation may be a useful therapeutic approach for chronic cerebrovascular disease and the resulting white matter lesions.

Oxidized-LDL Induces Metabolic Dysfunction in Retinal Pigment Epithelial Cells.

Recently, mitochondrial dysfunction has gained attention as a causative factor in the pathogenesis and progression of age-related macular degeneration (AMD). Mitochondrial damage plays a key role in metabolism and disrupts the balance of intracellular metabolic pathways, such as oxidative phosphorylation (OXPHOS) and glycolysis. In this study, we focused on oxidized low-density lipoprotein (ox-LDL), a major constituent of drusen that accumulates in the retina of patients with AMD, and investigated whether it could be a causative factor for metabolic alterations in retinal pigment epithelial (RPE) cells. We found that prolonged exposure to ox-LDL induced changes in fatty acid ß-oxidation (FAO), OXPHOS, and glycolytic activity and increased the mitochondrial reactive oxygen species production in RPE cells. Notably, the effects on metabolic alterations varied with the concentration and duration of ox-LDL treatment. In addition, we addressed the limitations of using ARPE-19 cells for retinal disease research by highlighting their lower barrier function and FAO activity compared to those of induced pluripotent stem cell-derived RPE cells. Our findings can aid in the elucidation of mechanisms underlying the metabolic alterations in AMD.

Slowly progressive cell death induced by GPx4-deficiency occurs via MEK1/ERK2 activation as a downstream signal after iron-independent lipid peroxidation.

Glutathione peroxidase 4 (GPx4) is an antioxidant enzyme that reduces phospholipid hydroperoxide. Studies have reported that the loss of GPx4 activity through anticancer drugs leads to ferroptosis, an iron-dependent lipid peroxidation-induced cell death. In this study, we established Tamoxifen-inducible GPx4-deficient Mouse embryonic fibroblast (MEF) cells (ETK1 cells) and found that Tamoxifen-inducible gene disruption of GPx4 induces slow cell death at ~72 h. In contrast, RSL3- or erastin-induced ferroptosis occurred quickly within 24 h. Therefore, we investigated the differences in these mechanisms between GPx4 gene disruption-induced cell death and RSL3- or erastin-induced ferroptosis. We found that GPx4-deficiency induced lipid peroxidation at 24 h in Tamoxifen-treated ETK1 cells, which was not suppressed by iron chelators, although lipid peroxidation in RSL3- or erastin-treated cells induced ferroptosis that was inhibited by iron chelators. We revealed that GPx4-deficient cell death was MEK1-dependent but RSL3- or erastin-induced ferroptosis was not, although MEK1/2 inhibitors suppressed both GPx4-deficient cell death and RSL3- or erastin-induced ferroptosis. In GPx4-deficient cell death, the phosphorylation of MEK1/2 and ERK2 was observed 39 h after lipid peroxidation, but ERK1 was not phosphorylated. Selective inhibitors of ERK2 inhibited GPx4-deficient cell death but not in RSL3- or erastin-induced cell death. These findings suggest that iron-independent lipid peroxidation due to GPx4 disruption induced cell death via the activation of MEK1/ERK2 as a downstream signal of lipid peroxidation in Tamoxifen-treated ETK1 cells. This indicates that GPx4 gene disruption induces slow cell death and involves a different pathway from RSL3- and erastin-induced ferroptosis in ETK1 cells.

Structural Analysis of Intracellular Lipid Radicals by LC/MS/MS Using a BODIPY-Based Profluorescent Nitroxide Probe.

Free radical-mediated lipid peroxidation (LPO) induces the formation of numerous lipid radicals, which contribute to the development of several oxidative diseases. To understand the mechanism of LPO in biological systems and the significance of these radicals, identifying the structures of individual lipid radicals is imperative. In this study, we developed an analytical method based on liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and a profluorescent nitroxide probe, N-(1-oxyl-2,2,6-trimethyl-6-pentylpiperidin-4-yl)-3-(5,5-difluoro-1,3-dimethyl-3H,5H-5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-7-yl)propanamide (BDP-Pen), for the detailed structural analysis of lipid radicals. The MS/MS spectra of BDP-Pen-lipid radical adducts showed product ions and thus allow the prediction of the lipid radical structures and individual detection of isomeric adducts. Using the developed technology, we separately detected the isomers of arachidonic acid (AA)-derived radicals generated in AA-treated HT1080 cells. This analytical system is a powerful tool for elucidating the mechanism of LPO in biological systems.

Preparation of N-2-Nitrophenylsulfenyl Imino Peptides and Their Catalyst-Controlled Diastereoselective Indolylation.

N-2-Nitrophenylsulfenyl imino dipeptides bearing various functional groups were successfully prepared by MnO2 -mediated oxidation and then subjected to diastereoselective indolylation. Each diastereomer of the adduct was selectively obtained from the same substrates using the appropriate chiral phosphoric acid catalysts. These transformations would be useful for synthesizing non-canonical amino acid-containing peptides as novel drug candidates.

Remote Electronic Effect of Chiral N-Heterocyclic Carbene Catalyst on an Asymmetric Benzoin Reaction.

A remote electronic effect of chiral aminoindanol-derived N-heterocyclic carbene catalyst on an asymmetric benzoin reaction was investigated. The catalyst bearing remote electron-withdrawing substituents increased enantioselectivity of the reaction at the cost of the reaction rate. DFT calculations rationalized the increased enantioselectivity.

Remote Electronic Tuning of Chiral N-Heterocyclic Carbenes.

Our recent efforts to develop novel N-Heterocyclic carbene (NHC)-catalyzed asymmetric reactions are described. During our investigation for development of the acylation reactions via acylazoliums generated by the reactions of NHCs and α-oxidized aldehydes, we have observed significant effects of substitution at a remote site of the carbene carbon of NHCs. In addition, we also observed a significant enhancement of the enantioselectivity by the addition of carboxylate anions. From this observation, we proposed a novel working hypothesis involving a formation of a complex of the substrate and additive to reinforce the recognition of the catalyst for enhancement of the catalytic performance of the asymmetric N-heterocyclic carbene system. By applying this concept, we achieved the kinetic resolutions of both cyclic and acyclic alcohols in excellent enantioselectivities. The effects of the remote substitution were also observed in intramolecular Stetter reaction and intermolecular benzoin reaction. In these reactions, the comparison of the catalytic performance of the NHCs bearing variable remote substitutions provided insights into the reaction mechanism because the remote substitution tuned the electronic nature of NHCs without affecting the steric and electrostatic factors around the reaction site. We also developed an intramolecular benzoin condensation involving two aldehydes, which is challenging to realize. Using the substrates bearing proper protecting groups, we succeeded in the stereo divergent synthesis of a variety of inososes, which are important intermediates for the synthesis of biologically active cyclitols.

Effects of selenium deficiency on biological results of X-ray and carbon-ion beam irradiation in mice.

The impact of radiation-induced hydrogen peroxide (H2O2) on the biological effects of X-rays and carbon-ion beams was investigated using a selenium-deficient (SeD) mouse model. Selenium is the active center of glutathione peroxidase (GSH-Px), and SeD mice lack the ability to degrade H2O2. Male and female SeD mice were prepared by feeding a torula yeast-based SeD diet and ultrapure water. Thirty-day survival rates after whole-body irradiation, radiation-induced leg contracture, and MRI-based redox imaging of the brain were assessed and compared between SeD and normal mice. Thirty-day lethality after whole-body 5.6 Gy irradiation with X-rays or carbon-ion beams was higher in the SeD mice than in the normal mice, while SeD did not give the notable difference between X-rays and carbon-ion beams. SeD also did not affect the maximum leg contracture level after irradiation with carbon-ion beams, but delayed the leg contraction rate. In addition, no marked effects of SeD were observed on variations in the redox status of the brain after irradiation. Collectively, the present results indicate that SeD slightly altered the biological effects of X-rays and/or carbon-ion beams. GSH-Px processes endogenous H2O2 generated through mitochondrial respiration, but does not have the capacity to degrade H2O2 produced by irradiation.

Ethoxyquin is a Competent Radical-Trapping Antioxidant for Preventing Ferroptosis in Doxorubicin Cardiotoxicity.

ABSTRACT: Doxorubicin (DOX) is an effective anti-cancer agent for various malignancies. Nevertheless, it has a side effect of cardiotoxicity, referred to as doxorubicin-induced cardiomyopathy (DIC), that is associated with a poorer prognosis. This cardiotoxicity limits the clinical use of DOX as a therapeutic agent for malignancies. Recently, ferroptosis, a form of regulated cell death induced by the accumulation of lipid peroxides, has been recognized as a major pathophysiology of DIC. Ethoxyquin is a lipophilic antioxidant widely used for food preservation and thus may be a potential therapeutic drug for preventing DIC. However, the efficacy of ethoxyquin against ferroptosis and DIC remains to be fully elucidated. Here, we investigated the inhibitory action of ethoxyquin against GPx4-deficient ferroptosis and its therapeutic efficacy against DOX-induced cell death in cultured cardiomyocytes and cardiotoxicity in a murine model of DIC. In cultured cardiomyocytes, ethoxyquin treatment effectively prevented GPx4-deficient ferroptosis. Ethoxyquin also prevented DOX-induced cell death, accompanied by the suppression of malondialdehyde (MDA) and mitochondrial lipid peroxides, which were induced by DOX. Furthermore, ethoxyquin significantly prevented DOX-induced cell death without any suppression of caspase cleavages representing apoptosis. In DIC mice, ethoxyquin treatment ameliorated cardiac impairments, such as contractile dysfunction and myocardial atrophy, and lung congestion. Ethoxyquin also suppressed serum lactate dehydrogenase and creatine kinase activities, decreased the levels of lipid peroxides such as MDA and acrolein, inhibited cardiac fibrosis, and reduced TUNEL-positive cells in the hearts of DIC mice. Collectively, ethoxyquin is a competent antioxidant for preventing ferroptosis in DIC and can be its prospective therapeutic drug.

Push-Pull Bisnaphthyridylamine Supramolecular Nanoparticles: Polarity-Induced Aggregation and Crystallization-Induced Emission Enhancement and Fluorescence Resonance Energy Transfer.

Emissive push-pull-type bisnaphthyridylamine derivatives (BNA-X: X=Me, Et, Bzl, Ph, BuBr, and BuTEMPO) aggregate in aqueous methanol. Furthermore, a two-step emission and aggregation process is controllable by varying the methanol-to-water ratio. At 2:3 MeOH/H2 O, crystallization-induced emission enhancement (CIEE) occurs via formation of an emissive crystal phase, whereas, at 1:9 MeOH/H2 O, aggregation-induced emission enhancement (AIEE) occurs, induced by emissive supramolecular nanoparticles (NPs). For BNA-Ph, the emission quantum yield was 25 times higher in aqueous methanol than that in pure methanol. Despite the high hydrophobicity of BNA-X (C log P=6.1-8.0), the spherical NPs were monodisperse (polydispersity indices <0.2). Moreover, the emissive NPs exhibited fluorescence resonance energy transfer (FRET) with pyrene; however, for BNA-X bearing the TEMPO radical (BNA-BuTEMPO), no FRET was observed because of quenching. In particular, the BNA-BuTEMPO NPs have a slow rotational correlation time (1.3 ns), suggesting applications as magnetic resonance imaging contrast agents with large relaxivity.

Drugs Repurposed as Antiferroptosis Agents Suppress Organ Damage, Including AKI, by Functioning as Lipid Peroxyl Radical Scavengers.

BACKGROUND: Ferroptosis, nonapoptotic cell death mediated by free radical reactions and driven by the oxidative degradation of lipids, is a therapeutic target because of its role in organ damage, including AKI. Ferroptosis-causing radicals that are targeted by ferroptosis suppressors have not been unequivocally identified. Because certain cytochrome P450 substrate drugs can prevent lipid peroxidation via obscure mechanisms, we evaluated their antiferroptotic potential and used them to identify ferroptosis-causing radicals. METHODS: Using a cell-based assay, we screened cytochrome P450 substrate compounds to identify drugs with antiferroptotic activity and investigated the underlying mechanism. To evaluate radical-scavenging activity, we used electron paramagnetic resonance-spin trapping methods and a fluorescence probe for lipid radicals, NBD-Pen, that we had developed. We then assessed the therapeutic potency of these drugs in mouse models of cisplatin-induced AKI and LPS/galactosamine-induced liver injury. RESULTS: We identified various US Food and Drug Administration-approved drugs and hormones that have antiferroptotic properties, including rifampicin, promethazine, omeprazole, indole-3-carbinol, carvedilol, propranolol, estradiol, and thyroid hormones. The antiferroptotic drug effects were closely associated with the scavenging of lipid peroxyl radicals but not significantly related to interactions with other radicals. The elevated lipid peroxyl radical levels were associated with ferroptosis onset, and known ferroptosis suppressors, such as ferrostatin-1, also functioned as lipid peroxyl radical scavengers. The drugs exerted antiferroptotic activities in various cell types, including tubules, podocytes, and renal fibroblasts. Moreover, in mice, the drugs ameliorated AKI and liver injury, with suppression of tissue lipid peroxidation and decreased cell death. CONCLUSIONS: Although elevated lipid peroxyl radical levels can trigger ferroptosis onset, some drugs that scavenge lipid peroxyl radicals can help control ferroptosis-related disorders, including AKI.

2-Oxo-histidine-containing dipeptides are functional oxidation products.

Imidazole-containing dipeptides (IDPs), such as carnosine and anserine, are found exclusively in various animal tissues, especially in the skeletal muscles and nerves. IDPs have antioxidant activity because of their metal-chelating and free radical-scavenging properties. However, the underlying mechanisms that would fully explain IDP antioxidant effects remain obscure. Here, using HPLC-electrospray ionization-tandem MS analyses, we comprehensively investigated carnosine and its related small peptides in the soluble fractions of mouse tissue homogenates and ubiquitously detected 2-oxo-histidine-containing dipeptides (2-oxo-IDPs) in all examined tissues. We noted enhanced production of the 2-oxo-IDPs in the brain of a mouse model of sepsis-associated encephalopathy. Moreover, in SH-SY5Y human neuroblastoma cells stably expressing carnosine synthase, H2O2 exposure resulted in the intracellular production of 2-oxo-carnosine, which was associated with significant inhibition of the H2O2 cytotoxicity. Notably, 2-oxo-carnosine showed a better antioxidant activity than endogenous antioxidants such as GSH and ascorbate. Mechanistic studies indicated that carnosine monooxygenation is mediated through the formation of a histidyl-imidazole radical, followed by the addition of molecular oxygen. Our findings reveal that 2-oxo-IDPs are metal-catalyzed oxidation products present in vivo and provide a revised paradigm for understanding the antioxidant effects of the IDPs.

Helical Nanographenes Embedded with Contiguous Azulene Units.

The azulene moiety, composed of contiguous pentagonal and heptagonal rings, is a structural defect that alters the electronic, magnetic, and structural properties of graphenes and graphene nanoribbons. However, nanographenes embedded with an azulene cluster have not been widely investigated because these compounds are difficult to synthesize in their pure form. Herein, azulene-embedded nanographenes bearing a unique cove-type edge were synthesized by a novel synthetic protocol. Experimental and theoretical investigations revealed that this cove edge imparts stable helical chirality, unlike normal cove edges. The in-solution self-association behavior and the structural, electronic, and electrochemical properties were also described in detail.

Method for Structural Determination of Lipid-Derived Radicals.

Diversified oxidized-lipid molecules are responsible for inflammation and cell death, including ferroptosis. Lipid radicals are the source of these oxidized lipids, which are the initial key molecules in the lipid peroxidation chain reaction. However, owing to their extremely high reactivity and short half-life, an established detection technique is not available. Here, we propose a high-performance liquid chromatography fluorometry and high-resolution tandem mass spectrometry system combined with a fluorescent probe as a structural analysis method for lipid-derived radicals. We detected 132 lipid-derived radicals, including 111 new species, from five polyunsaturated fatty acids. In addition, a database was constructed for which the initial fatty acid could be determined using the radical structure. Further, 12 endogenous lipid-derived radicals were identified in carcinogen-induced liver cancer mouse models. Therefore, this method and its corresponding database will provide novel insights into mechanisms underlying the lipid peroxidation, including the associated inflammation and ferroptosis.

Ascorbic acid and CoQ10 ameliorate the reproductive ability of superoxide dismutase 1-deficient female mice†.

Superoxide dismutase 1 suppresses oxidative stress within cells by decreasing the levels of superoxide anions. A dysfunction of the ovary and/or an aberrant production of sex hormones are suspected causes for infertility in superoxide dismutase 1-knockout mice. We report on attempts to rescue the infertility in female knockout mice by providing two antioxidants, ascorbic acid and/or coenzyme Q10, as supplements in the drinking water of the knockout mice after weaning and on an investigation of their reproductive ability. On the first parturition, 80% of the untreated knockout mice produced smaller litter sizes compared with wild-type mice (average 2.8 vs 7.3 pups/mouse), and supplementing with these antioxidants failed to improve these litter sizes. However, in the second parturition of the knockout mice, the parturition rate was increased from 18% to 44-75% as the result of the administration of antioxidants. While plasma levels of progesterone at 7.5 days of pregnancy were essentially the same between the wild-type and knockout mice and were not changed by the supplementation of these antioxidants, sizes of corpus luteum cells, which were smaller in the knockout mouse ovaries after the first parturition, were significantly ameliorated in the knockout mouse with the administration of the antioxidants. Moreover, the impaired vasculogenesis in uterus/placenta was also improved by ascorbic acid supplementation. We thus conclude that ascorbic acid and/or coenzyme Q10 are involved in maintaining ovarian and uterus/placenta homeostasis against insults that are augmented during pregnancy and that their use might have positive effects in terms of improving female fertility.

Characterization and Water-Proton Longitudinal Relaxivities of Liposome-Type Radical Nanoparticles Prepared via a Supramolecular Approach.

For the construction of metal-free magnetic resonance imaging (MRI) contrast agents, radical-based nanoparticles (RNPs) are promising materials because they allow the water-proton longitudinal relaxivity (r1) to be enhanced not only by paramagnetic resonance effects but also by prolonging the rotational correlation times (τR). However, the τR effect is limited because the radical units are often located within the central hydrophobic core of oil-in-water (o/w) emulsions, resulting in a lack of water molecules surrounding the radical units. In this study, to construct supramolecular RNPs that have high r1 values, we designed a liposome-type RNP in which the radical units are located at positions with sufficient surrounding water molecules. Using this strategy, PRO1 with a PROXYL framework was prepared by introducing hydrophilic groups on both sides of the radical unit. The RNP composed of PRO1 formed spherical nanoparticles approximately 100 nm in size and yielded a higher r1 value (0.26 mM-1 s-1) compared to those of small radical species and similar supramolecular o/w emulsion-type nanoparticles (0.17 mM-1 s-1 in PRO2).

Asymmetric Synthesis of α-Amino Phosphonic Acids using Stable Imino Phosphonate as a Universal Precursor.

A practical method for synthesizing chiral α-amino phosphonic acid derivatives was developed. Readily available and stable N-o-nitrophenylsulfenyl (Nps) imino phosphonate was utilized as a substrate for a highly enantioselective Friedel-Crafts-type addition of indole or pyrrole nucleophiles catalyzed by chiral phosphoric acid. The resulting adduct was easily converted into N-9-fluorenylmethyloxycarbonyl (Fmoc) amino phosphonic acid, which is useful for synthesizing peptides containing an amino phosphonic acid.