Pub Date : 2025-10-09DOI: 10.1016/j.rbc.2025.100061
Valerie B. O'Donnell , Valery Bochkov
Oxidized phospholipids (oxPL) are generated by enzymatic or non-enzymatic reactions and play diverse roles in immunity and inflammation. OxPL are elevated in tissues from many human diseases and are now recognized as endogenous damage-associated molecular patterns (DAMPs) that alert the immune system to challenge. Early studies focused on the role(s) of non-enzymatically-formed oxPCs in cardiovascular disease, while more recently, the controlled generation of enzymatically-oxidized PL (eoxPL) in blood cells and their participation in physiological hemostasis has been delineated. In the last decade, there has been an explosion of research into their formation and roles in ferroptosis, a form of cell death driven by iron and lipid oxidation. This mini review aims to bring the reader up to date with recent work in this area, focused on discoveries over the last few years that firmly extend our knowledge of the roles of oxPL as mediators of ferroptosis, innate and adaptive immunity.
{"title":"Oxidized phospholipids in ferroptosis, immunity and inflammation","authors":"Valerie B. O'Donnell , Valery Bochkov","doi":"10.1016/j.rbc.2025.100061","DOIUrl":"10.1016/j.rbc.2025.100061","url":null,"abstract":"<div><div>Oxidized phospholipids (oxPL) are generated by enzymatic or non-enzymatic reactions and play diverse roles in immunity and inflammation. OxPL are elevated in tissues from many human diseases and are now recognized as endogenous damage-associated molecular patterns (DAMPs) that alert the immune system to challenge. Early studies focused on the role(s) of non-enzymatically-formed oxPCs in cardiovascular disease, while more recently, the controlled generation of enzymatically-oxidized PL (eoxPL) in blood cells and their participation in physiological hemostasis has been delineated. In the last decade, there has been an explosion of research into their formation and roles in ferroptosis, a form of cell death driven by iron and lipid oxidation. This mini review aims to bring the reader up to date with recent work in this area, focused on discoveries over the last few years that firmly extend our knowledge of the roles of oxPL as mediators of ferroptosis, innate and adaptive immunity.</div></div>","PeriodicalId":101065,"journal":{"name":"Redox Biochemistry and Chemistry","volume":"14 ","pages":"Article 100061"},"PeriodicalIF":0.0,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145268187","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-12DOI: 10.1016/j.rbc.2025.100060
Claudia Cecilia Vera , Jesús M.N. Morales , María del Pilar Guauque Torres , Mariana P. Serrano , Claudio D. Borsarelli
A photosensitized oxidative crosslinking of proteins (POCP) reaction was applied in air-saturated phosphate buffer solutions of bovine serum albumin (BSA) to obtain soluble protein nanoparticles of approximately 100 nm in diameter. A royal blue LED was used as the excitation source for the photosensitizer molecule ruthenium (II) tris(2,2′-bipyridyl) dication (Ru(bpy)32+), in the presence of the electron acceptor persulfate anion (S2O82−). The redox quenching products prompted the formation of side-chain tyrosyl radicals, which served as intermediaries in the covalent attachment between proteins, leading to the formation of dityrosine (Tyr2) links. However, the dissolved oxygen competes efficiently with S2O82− to quench the excited photosensitizer, thereby generating singlet molecular oxygen (1O2), which reacts with electron-rich protein residues, which in turn induce an additional oxidative pattern of BSA. Consequently, under air-saturated conditions, the POCP gives rise to a series of oxygen-dependent and -independent reactions, resulting in the protein crosslinking with oxidative modifications. The esterase-like activity efficacy of BSA oxidized solely by 1O2 and after the formation of oligomeric protein nanoparticles by POCP was reduced by 51 % and 73 %, respectively, as compared with that of the native BSA. The combination of the oxidative degradation of key residues in the active sites and steric impediment due to protein oligomerization was found to be associated with this result.
{"title":"Photosensitized oxidative crosslinking of bovine serum albumin and the impact on its esterase-like activity","authors":"Claudia Cecilia Vera , Jesús M.N. Morales , María del Pilar Guauque Torres , Mariana P. Serrano , Claudio D. Borsarelli","doi":"10.1016/j.rbc.2025.100060","DOIUrl":"10.1016/j.rbc.2025.100060","url":null,"abstract":"<div><div>A photosensitized oxidative crosslinking of proteins (POCP) reaction was applied in air-saturated phosphate buffer solutions of bovine serum albumin (BSA) to obtain soluble protein nanoparticles of approximately 100 nm in diameter. A royal blue LED was used as the excitation source for the photosensitizer molecule ruthenium (II) tris(2,2′-bipyridyl) dication (Ru(bpy)<sub>3</sub><sup>2+</sup>), in the presence of the electron acceptor persulfate anion (S<sub>2</sub>O<sub>8</sub><sup>2−</sup>). The redox quenching products prompted the formation of side-chain tyrosyl radicals, which served as intermediaries in the covalent attachment between proteins, leading to the formation of dityrosine (Tyr<sub>2</sub>) links. However, the dissolved oxygen competes efficiently with S<sub>2</sub>O<sub>8</sub><sup>2−</sup> to quench the excited photosensitizer, thereby generating singlet molecular oxygen (<sup>1</sup>O<sub>2</sub>), which reacts with electron-rich protein residues, which in turn induce an additional oxidative pattern of BSA. Consequently, under air-saturated conditions, the POCP gives rise to a series of oxygen-dependent and -independent reactions, resulting in the protein crosslinking with oxidative modifications. The esterase-like activity efficacy of BSA oxidized solely by <sup>1</sup>O<sub>2</sub> and after the formation of oligomeric protein nanoparticles by POCP was reduced by 51 % and 73 %, respectively, as compared with that of the native BSA. The combination of the oxidative degradation of key residues in the active sites and steric impediment due to protein oligomerization was found to be associated with this result.</div></div>","PeriodicalId":101065,"journal":{"name":"Redox Biochemistry and Chemistry","volume":"14 ","pages":"Article 100060"},"PeriodicalIF":0.0,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145107888","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-19DOI: 10.1016/j.rbc.2025.100058
Noelle Reimers, Libin Xu
Ferroptosis is a type of regulated cell death that is dependent on iron and driven by lipid peroxidation. Polyunsaturated fatty acids (PUFAs) sensitize cells to ferroptosis as they are prone to lipid peroxidation while monounsaturated fatty acids confer resistance to ferroptosis when incorporated into the lipid membrane as they are much less reactive toward lipid peroxidation. Recently, in addition to fatty acid-derived lipids, isoprenoid-derived lipids have been found to regulate ferroptosis. Specifically, ferroptosis suppressor protein 1 (FSP1) was found to be anti-ferroptotic as it reduces the oxidized forms of coenzyme Q10 and vitamin K to their reduced quinol forms, which are phenolic radical-trapping antioxidants. Vitamins D3 and A have also been found to inhibit ferroptosis in cancer cells. Furthermore, it has been shown that metabolites along the cholesterol synthesis pathway, including squalene, cholesterol, desmosterol, and 7-dehydrocholesterol (7-DHC), can protect cells against ferroptosis in vitro. Despite large variations in the reactivities of these lipids toward lipid peroxidation, they generally exhibit anti-ferroptotic properties. In this review, we will discuss the peroxidation rate constants and mechanisms of these isoprenoid-derived lipids and how they might contribute to their roles in ferroptosis.
{"title":"Peroxidation rate constants and mechanisms of isoprenoid-derived lipids and their roles in ferroptosis","authors":"Noelle Reimers, Libin Xu","doi":"10.1016/j.rbc.2025.100058","DOIUrl":"10.1016/j.rbc.2025.100058","url":null,"abstract":"<div><div>Ferroptosis is a type of regulated cell death that is dependent on iron and driven by lipid peroxidation. Polyunsaturated fatty acids (PUFAs) sensitize cells to ferroptosis as they are prone to lipid peroxidation while monounsaturated fatty acids confer resistance to ferroptosis when incorporated into the lipid membrane as they are much less reactive toward lipid peroxidation. Recently, in addition to fatty acid-derived lipids, isoprenoid-derived lipids have been found to regulate ferroptosis. Specifically, ferroptosis suppressor protein 1 (FSP1) was found to be anti-ferroptotic as it reduces the oxidized forms of coenzyme Q<sub>10</sub> and vitamin K to their reduced quinol forms, which are phenolic radical-trapping antioxidants. Vitamins D<sub>3</sub> and A have also been found to inhibit ferroptosis in cancer cells. Furthermore, it has been shown that metabolites along the cholesterol synthesis pathway, including squalene, cholesterol, desmosterol, and 7-dehydrocholesterol (7-DHC), can protect cells against ferroptosis in vitro. Despite large variations in the reactivities of these lipids toward lipid peroxidation, they generally exhibit anti-ferroptotic properties. In this review, we will discuss the peroxidation rate constants and mechanisms of these isoprenoid-derived lipids and how they might contribute to their roles in ferroptosis.</div></div>","PeriodicalId":101065,"journal":{"name":"Redox Biochemistry and Chemistry","volume":"13 ","pages":"Article 100058"},"PeriodicalIF":0.0,"publicationDate":"2025-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144890329","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-29DOI: 10.1016/j.rbc.2025.100057
Lawrence J. Marnett
Prostaglandins are an important class of bioactive lipids derived from arachidonic acid. The different classes of prostaglandins are produced from a common intermediate, called prostaglandin H2. Prostaglandin H2 is produced by reduction of the initial oxygenation product, prostaglandin G2, which contains cyclic peroxide and hydroperoxide functionalities. Extensive experimentation has established that arachidonic acid is oxygenated to prostaglandin G2 via a free radical mechanism. This review describes the key steps in the oxygenation of arachidonic acid and the evidence that supports the most widely accepted mechanism.
{"title":"The chemistry of arachidonic acid oxygenation by prostaglandin endoperoxide synthase","authors":"Lawrence J. Marnett","doi":"10.1016/j.rbc.2025.100057","DOIUrl":"10.1016/j.rbc.2025.100057","url":null,"abstract":"<div><div>Prostaglandins are an important class of bioactive lipids derived from arachidonic acid. The different classes of prostaglandins are produced from a common intermediate, called prostaglandin H<sub>2</sub>. Prostaglandin H<sub>2</sub> is produced by reduction of the initial oxygenation product, prostaglandin G<sub>2</sub>, which contains cyclic peroxide and hydroperoxide functionalities. Extensive experimentation has established that arachidonic acid is oxygenated to prostaglandin G<sub>2</sub> via a free radical mechanism. This review describes the key steps in the oxygenation of arachidonic acid and the evidence that supports the most widely accepted mechanism.</div></div>","PeriodicalId":101065,"journal":{"name":"Redox Biochemistry and Chemistry","volume":"13 ","pages":"Article 100057"},"PeriodicalIF":0.0,"publicationDate":"2025-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144522829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-19DOI: 10.1016/j.rbc.2025.100056
Ryotaro Kano , Hideki Shirakawa , David C. Poole , Daisuke Hoshino , Yutaka Kano
Evaluation of the effects of muscle contractions on reactive oxygen species (ROS) concentrations and their intracellular and intraorganelle dynamics is important for understanding protection against cellular damage and resolving the mechanistic bases for muscle plasticity after training, with aging and in disease. Because of the highly reactive nature of ROS, measurement methods have typically been limited to a few established approaches. In this review, we discuss the advantages and limitations of the typical methods for detecting ROS in skeletal muscle to date. In particular, we focus on the importance of in vivo imaging using ROS-sensitive genetically encoded fluorescent proteins. The pressing need for quantitative analysis of each organelle, such as mitochondria, to advance our comprehensive biological understanding of ROS dynamics during muscle contractions is stressed. These considerations provide a direction for more fully understanding exercise-induced redox signaling.
{"title":"State of the art in vivo reactive oxygen species measurements in skeletal muscle using fluorescent proteins","authors":"Ryotaro Kano , Hideki Shirakawa , David C. Poole , Daisuke Hoshino , Yutaka Kano","doi":"10.1016/j.rbc.2025.100056","DOIUrl":"10.1016/j.rbc.2025.100056","url":null,"abstract":"<div><div>Evaluation of the effects of muscle contractions on reactive oxygen species (ROS) concentrations and their intracellular and intraorganelle dynamics is important for understanding protection against cellular damage and resolving the mechanistic bases for muscle plasticity after training, with aging and in disease. Because of the highly reactive nature of ROS, measurement methods have typically been limited to a few established approaches. In this review, we discuss the advantages and limitations of the typical methods for detecting ROS in skeletal muscle to date. In particular, we focus on the importance of <em>in vivo</em> imaging using ROS-sensitive genetically encoded fluorescent proteins. The pressing need for quantitative analysis of each organelle, such as mitochondria, to advance our comprehensive biological understanding of ROS dynamics during muscle contractions is stressed. These considerations provide a direction for more fully understanding exercise-induced redox signaling.</div></div>","PeriodicalId":101065,"journal":{"name":"Redox Biochemistry and Chemistry","volume":"13 ","pages":"Article 100056"},"PeriodicalIF":0.0,"publicationDate":"2025-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144471120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-01DOI: 10.1016/j.rbc.2025.100054
Ned A. Porter
The chemical framework for free radical chain oxidation of naturally-occurring lipids, commonly referred to as peroxidation, has provided a basis for understanding important processes in biology. H-atom transfer to peroxyl free radicals and olefin addition of those radicals are the primary rate-determining steps in peroxidation, but the lipid carbon radicals generated in these primary steps have multiple mechanistic pathways available. Oxygen addition, homolytic intramolecular substitution (sHi) and various cyclization reactions of intermediate peroxyl and alkoxyl radicals leads to a diverse set of products from polyunsaturated fatty acids and phospholipid esters. 5,7-Diene sterols are particularly reactive H-atom donors and give rise to complex product mixtures. The mechanistic guidelines for the important transformations of lipid peroxidation are summarized here and the connection between these fundamental chemical conversions and important enzymatic and non-enzymatic biological processes are outlined.
{"title":"Chemical mechanisms of lipid peroxidation","authors":"Ned A. Porter","doi":"10.1016/j.rbc.2025.100054","DOIUrl":"10.1016/j.rbc.2025.100054","url":null,"abstract":"<div><div>The chemical framework for free radical chain oxidation of naturally-occurring lipids, commonly referred to as peroxidation, has provided a basis for understanding important processes in biology. H-atom transfer to peroxyl free radicals and olefin addition of those radicals are the primary rate-determining steps in peroxidation, but the lipid carbon radicals generated in these primary steps have multiple mechanistic pathways available. Oxygen addition, homolytic intramolecular substitution (<em>s</em><sub><em>H</em></sub><em>i</em>) and various cyclization reactions of intermediate peroxyl and alkoxyl radicals leads to a diverse set of products from polyunsaturated fatty acids and phospholipid esters. 5,7-Diene sterols are particularly reactive H-atom donors and give rise to complex product mixtures. The mechanistic guidelines for the important transformations of lipid peroxidation are summarized here and the connection between these fundamental chemical conversions and important enzymatic and non-enzymatic biological processes are outlined.</div></div>","PeriodicalId":101065,"journal":{"name":"Redox Biochemistry and Chemistry","volume":"12 ","pages":"Article 100054"},"PeriodicalIF":0.0,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144178655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-06-01DOI: 10.1016/j.rbc.2025.100053
Martina Paumann-Page , Louisa V. Ashby , Irada Khalilova , Nicholas J. Magon , Stefan Hofbauer , Louise N. Paton , Paul G. Furtmüller , Christian Obinger , Anthony J. Kettle
{"title":"Corrigendum to “Hypochlorous acid inactivates myeloperoxidase inside phagocytosing neutrophils” [Redox Biochem. Chem. 5–6 (2023) 100008]","authors":"Martina Paumann-Page , Louisa V. Ashby , Irada Khalilova , Nicholas J. Magon , Stefan Hofbauer , Louise N. Paton , Paul G. Furtmüller , Christian Obinger , Anthony J. Kettle","doi":"10.1016/j.rbc.2025.100053","DOIUrl":"10.1016/j.rbc.2025.100053","url":null,"abstract":"","PeriodicalId":101065,"journal":{"name":"Redox Biochemistry and Chemistry","volume":"12 ","pages":"Article 100053"},"PeriodicalIF":0.0,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144253541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-05-05DOI: 10.1016/j.rbc.2025.100052
Michael J. Davies
Proteins are highly abundant and readily oxidized targets of reactive species formed in biological systems, with these often accounting for greater than 50 % of the dry mass of biological samples. Of the amino acids present in proteins, the sulfur-containing amino acids cysteine (Cys), cystine and methionine (Met) are some of the most reactive species with a range of biologically-relevant modifying agents including radicals, two-electron species and also many electrophiles. Reaction with Cys gives a wide range of both reversible and irreversible species. Lesser numbers of products are well-characterized for cystine and Met. For the latter, the sulfoxide is often the most abundant product, but other species including the cyclic species dehydromethionine, and methionine sulfone have been characterized and shown to be major species under some circumstances. Whilst the sulfone has been widely reported to arise from the sulfoxide as a result of further oxidation, increasing evidence suggests that it can also be formed directly, without the intermediacy of the sulfoxide, and particularly with singlet oxygen (1O2). Whilst the sulfoxide is subject to reduction (e.g. via methionine sulfoxide reductases) and further metabolism in vivo, the sulfone appears to be a stable product and may therefore under certain circumstances be a biomarker of Met oxidation. This article briefly reviews the oxidation chemistry of Cys and cystine, and a more detailed discussion of the mechanisms of Met oxidation, formation of the sulfoxide, dehydromethionine and sulfone, and the biological fates and activities of these species.
{"title":"Methionine oxidation products as biomarkers of oxidative damage to proteins and modulators of cellular metabolism and toxicity","authors":"Michael J. Davies","doi":"10.1016/j.rbc.2025.100052","DOIUrl":"10.1016/j.rbc.2025.100052","url":null,"abstract":"<div><div>Proteins are highly abundant and readily oxidized targets of reactive species formed in biological systems, with these often accounting for greater than 50 % of the dry mass of biological samples. Of the amino acids present in proteins, the sulfur-containing amino acids cysteine (Cys), cystine and methionine (Met) are some of the most reactive species with a range of biologically-relevant modifying agents including radicals, two-electron species and also many electrophiles. Reaction with Cys gives a wide range of both reversible and irreversible species. Lesser numbers of products are well-characterized for cystine and Met. For the latter, the sulfoxide is often the most abundant product, but other species including the cyclic species dehydromethionine, and methionine sulfone have been characterized and shown to be major species under some circumstances. Whilst the sulfone has been widely reported to arise from the sulfoxide as a result of further oxidation, increasing evidence suggests that it can also be formed directly, without the intermediacy of the sulfoxide, and particularly with singlet oxygen (<sup>1</sup>O<sub>2</sub>). Whilst the sulfoxide is subject to reduction (e.g. via methionine sulfoxide reductases) and further metabolism <em>in vivo</em>, the sulfone appears to be a stable product and may therefore under certain circumstances be a biomarker of Met oxidation. This article briefly reviews the oxidation chemistry of Cys and cystine, and a more detailed discussion of the mechanisms of Met oxidation, formation of the sulfoxide, dehydromethionine and sulfone, and the biological fates and activities of these species.</div></div>","PeriodicalId":101065,"journal":{"name":"Redox Biochemistry and Chemistry","volume":"12 ","pages":"Article 100052"},"PeriodicalIF":0.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143912905","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-04-15DOI: 10.1016/j.rbc.2025.100051
Huan Liu , Lusine Tonoyan , Béla Reiz , Arno G. Siraki
Amplex Red (AR) is commonly used to detect extracellular hydrogen peroxide (H2O2) and is considered a cell-impermeable compound. However, it would appear capable of entering cells based on its phenoxazine substructure and the report of its mitochondrial membrane permeability. Additionally, myeloperoxidase (MPO) oxidation of AR produces a fluorescent compound, resorufin, which has been reported, though the mechanism is not well-studied. EPR spin trapping using glutathione (GSH) revealed that AR metabolism produced AR radicals and glutathionyl radicals (GS•). An intermediate metabolite, 3,7-dihydroxyphenoxazine, was observed by liquid chromatography-mass spectrometry (LC-MS), which supported AR radical disproportionation first and subsequently N-oxidation. Besides, in the presence of GSH, the formation of resorufin decreased significantly evidencing the reactivity of radical intermediates. Three types of AR-GS adduct were found using LC-MS and the resorufin GS-adduct was the dominant one. Regarding intracellular findings in HL-60 cells (that highly express MPO), LC-MS and fluorescence analysis showed AR penetrated the cell membrane and was oxidized by cellular MPO. Interestingly, we demonstrated that the oxidation of AR in HL-60 cells showed a significant time dependence; PF-1355, an MPO inhibitor, inhibited the oxidation of AR by MPO. Cell viability (ATP) revealed that 200 μM AR significantly decreased viability in HL-60 cells in 6 h. We also found that AR-mediated decreased total GSH and increased protein-radical formation. These findings revealed that AR is cell-permeable, and AR radicals induce cellular oxidative distress and lead to the formation of protein radicals, which correlate with the MPO-mediated mechanism of cytotoxicity.
Amplex Red （AR）通常用于检测细胞外过氧化氢（H2O2），被认为是一种细胞不渗透的化合物。然而，根据其苯恶嗪亚结构和线粒体膜通透性的报道，它似乎能够进入细胞。此外，髓过氧化物酶（MPO）氧化AR会产生一种荧光化合物，再间苯磺酸，这是有报道的，但其机制尚未得到很好的研究。利用谷胱甘肽（GSH）捕获EPR自旋表明，AR代谢产生AR自由基和谷胱甘肽基自由基（GS•）。液相色谱-质谱联用（LC-MS）观察到中间代谢物3,7-二羟基苯恶嗪（3,7-dihydroxyphenoxazine）首先支持AR自由基歧化，然后支持n -氧化。此外，在GSH的存在下，再间酚的形成明显减少，证明自由基中间体的反应性。LC-MS分析发现了3种类型的AR-GS加合物，其中再间酚类gs加合物占主导地位。对于高表达MPO的HL-60细胞，LC-MS和荧光分析显示AR穿透细胞膜并被细胞MPO氧化。有趣的是，我们证明了HL-60细胞中AR的氧化表现出显著的时间依赖性；MPO抑制剂PF-1355抑制了MPO对AR的氧化作用。细胞活力（ATP）显示，200 μM AR在6小时内显著降低HL-60细胞的活力。我们还发现AR介导的总GSH降低和蛋白自由基形成增加。这些发现表明，AR具有细胞渗透性，AR自由基诱导细胞氧化应激并导致蛋白自由基的形成，这与mpo介导的细胞毒性机制有关。
{"title":"Amplex Red cellular uptake produces radical intermediates by myeloperoxidase and mediates oxidative stress","authors":"Huan Liu , Lusine Tonoyan , Béla Reiz , Arno G. Siraki","doi":"10.1016/j.rbc.2025.100051","DOIUrl":"10.1016/j.rbc.2025.100051","url":null,"abstract":"<div><div>Amplex Red (AR) is commonly used to detect extracellular hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and is considered a cell-impermeable compound. However, it would appear capable of entering cells based on its phenoxazine substructure and the report of its mitochondrial membrane permeability. Additionally, myeloperoxidase (MPO) oxidation of AR produces a fluorescent compound, resorufin, which has been reported, though the mechanism is not well-studied. EPR spin trapping using glutathione (GSH) revealed that AR metabolism produced AR radicals and glutathionyl radicals (GS<sup>•</sup>). An intermediate metabolite, 3,7-dihydroxyphenoxazine, was observed by liquid chromatography-mass spectrometry (LC-MS), which supported AR radical disproportionation first and subsequently N-oxidation. Besides, in the presence of GSH, the formation of resorufin decreased significantly evidencing the reactivity of radical intermediates. Three types of AR-GS adduct were found using LC-MS and the resorufin GS-adduct was the dominant one. Regarding intracellular findings in HL-60 cells (that highly express MPO), LC-MS and fluorescence analysis showed AR penetrated the cell membrane and was oxidized by cellular MPO. Interestingly, we demonstrated that the oxidation of AR in HL-60 cells showed a significant time dependence; PF-1355, an MPO inhibitor, inhibited the oxidation of AR by MPO. Cell viability (ATP) revealed that 200 μM AR significantly decreased viability in HL-60 cells in 6 h. We also found that AR-mediated decreased total GSH and increased protein-radical formation. These findings revealed that AR is cell-permeable, and AR radicals induce cellular oxidative distress and lead to the formation of protein radicals, which correlate with the MPO-mediated mechanism of cytotoxicity.</div></div>","PeriodicalId":101065,"journal":{"name":"Redox Biochemistry and Chemistry","volume":"12 ","pages":"Article 100051"},"PeriodicalIF":0.0,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143851843","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-04-13DOI: 10.1016/j.rbc.2025.100050
M. Ignasiak , K.J. Frąckowiak , E. Fuentes-Lemus , P.M. Hägglund , L. Gamon , M.J. Davies , Ł. Marczak , B. Marciniak
3-carboxybenzophenone (CB) is an efficient photosensitizer that can oxidize multiple amino acid side chains in peptides and proteins via electron transfer (ET) reactions yielding various radicals and radical ions. Recombination reactions of these species can yield CBH-adducts and cross-links, whereas secondary reactions can give radicals on other side chains and further products. Prevention of initial radical formation, or interception of intermediate radicals is predicted to modulate the extent of protein damage. Consequently, in this work the effect of iodide ions (I‾) on CB-photosensitized oxidation was investigated with Trp and TyrOH, as these moieties are prone to photooxidation. A scavenging effect of I‾ on the formation of TyrO• radicals was readily detected in kinetic experiments using laser flash photolysis, whilst effects on TrpN• radical formation remain ambiguous (due to overlap of the absorptions of transient absorption spectra of TrpN• and CBH• radicals). Addition of I‾ suppresses oxidation of Trp and TyrOH, with this resulting in lower concentrations of di-Trp, di-Tyr and adducts with CBH, without formation of additional products involving I‾. The effect of I‾ was also analysed for a model protein – lysozyme – with a protective effect observed against loss of activity on illumination with CB. Multiple products were identified, including adducts of CBH to Trp, TyrOH or Met. The formation of crosslinks arising from CB-mediated photo-oxidation of lysozyme was limited in the presence of I‾. Together these data indicate that I‾ modulates photodamage induced by CB to peptide and protein targets.
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