Cold stress-induced ferroptosis involves the ASK1-p38 pathway.

A wide variety of cell death mechanisms, such as ferroptosis, have been proposed in mammalian cells, and the classification of cell death attracts global attention because each type of cell death has the potential to play causative roles in specific diseases. However, the precise molecular mechanisms leading to cell death are poorly understood, particularly in ferroptosis. Here, we show that continuous severe cold stress induces ferroptosis and the ASK1-p38 MAPK pathway in multiple cell lines. The activation of the ASK1-p38 pathway is mediated by critical determinants of ferroptosis: MEK activity, iron ions, and lipid peroxide. The chemical compound erastin, a potent ferroptosis inducer, also activates the ASK1-p38 axis downstream of lipid peroxide accumulation and leads to ASK1-dependent cell death in a cell type-specific manner. These lines of evidence provide mechanistic insight into ferroptosis, a type of regulated necrosis.

Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis.

Ferroptosis is form of regulated nonapoptotic cell death that is involved in diverse disease contexts. Small molecules that inhibit glutathione peroxidase 4 (GPX4), a phospholipid peroxidase, cause lethal accumulation of lipid peroxides and induce ferroptotic cell death. Although ferroptosis has been suggested to involve accumulation of reactive oxygen species (ROS) in lipid environments, the mediators and substrates of ROS generation and the pharmacological mechanism of GPX4 inhibition that generates ROS in lipid environments are unknown. We report here the mechanism of lipid peroxidation during ferroptosis, which involves phosphorylase kinase G2 (PHKG2) regulation of iron availability to lipoxygenase enzymes, which in turn drive ferroptosis through peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position; indeed, pretreating cells with PUFAs containing the heavy hydrogen isotope deuterium at the site of peroxidation (D-PUFA) prevented PUFA oxidation and blocked ferroptosis. We further found that ferroptosis inducers inhibit GPX4 by covalently targeting the active site selenocysteine, leading to accumulation of PUFA hydroperoxides. In summary, we found that PUFA oxidation by lipoxygenases via a PHKG2-dependent iron pool is necessary for ferroptosis and that the covalent inhibition of the catalytic selenocysteine in Gpx4 prevents elimination of PUFA hydroperoxides; these findings suggest new strategies for controlling ferroptosis in diverse contexts.

[Effect of cysteine lithium salt on lipid peroxidation in the model of dopaminergic system disorder].

The antioxidant activity of a newly synthesized compound, cysteine lithium salt, was discovered using methyl oleate oxidation model of dopaminergic system disorder. Experimental data show that injection of the synthesized compound in animals with experimentally induced L-DOPA brings to normalization effect conditioned by inhibiting free radical reactions in the nuclear and mitochondrial fractions of brain cells. Antioxidant effect of the synthesized compound is related to its chemical structure that contains cysteine--an agent involved in the biosynthesis of glutathione, which is a component of glutathione peroxidase and glutathione reductase enzymes participating in the antiradical protection system.

Deciphering the Molecular Details of the Lipoxin Formation Mechanism in the 5(S),15(S)-DiHpETE Biosynthetic Pathway Catalyzed by Reticulocyte 15-Lipoxygenase-1.

Chronic inflammation is now widely recognized to play important roles in many commonly occurring diseases, including COVID-19. The resolution response to this chronic inflammation is an active process governed by specialized pro-resolving mediators (SPMs) like the lipid mediators known as lipoxins. The biosynthesis of lipoxins is catalyzed by several lipoxygenases (LOXs) from arachidonic acid. However, the molecular details of the mechanisms involved are not well known yet. In this paper, we have combined molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations to analyze how reticulocyte 15-LOX-1 catalyzes the production of lipoxins from 5(S),15(S)-diHpETE. Our results indicate that the dehydration mechanism from 5(S),15(S)-diHpETE, via the formation of an epoxide, presents huge energy barriers even though it was one of the two a priori synthetic proposals. This result is compatible with the fact that no epoxide has been directly detected as an intermediate in the catalytic formation of lipoxins from 5(S),15(S)-diHpETE. Conversely, the oxygenation of 5(S),15(S)-diHpETE at C14 is feasible because there is an open channel connecting the protein surface with this carbon atom, and the energy barrier for oxygen addition through this channel is small. The analysis of the following steps of this mechanism, leading to the corresponding hydroperoxide at the 15-LOX-1 active site, indicates that the oxygenation mechanism will lead to the formation of lipoxinB4 after the final action of a reductase. In contrast, our calculations are in agreement with experiments that lipoxinA4 cannot derive from 5(S),15(S)-diHpETE by either of the two proposed mechanisms and that 5(S),15(S)-diHETE is not an intermediate of lipoxin biosynthesis catalyzed by 15-LOX-1.

trans-4-Hydroxy-2-hexenal: a reactive metabolite from the macrocyclic pyrrolizidine alkaloid senecionine.

The toxicity of macrocyclic pyrrolizidine alkaloids in the livers of man and animals has been attributed to the formation of reactive pyrroles from dihydropyrrolizines. Now a novel metabolite, trans-4-hydroxy-2-hexenal, has been isolated from the macrocyclic pyrrolizidine alkaloid senecionine, in an in vitro hepatic microsomal system. Other alkenals such as trans-4-hydroxy-2-nonenal have previously been isolated from microsomal systems when treated with halogenated hydrocarbons or subjected to lipid peroxidation. The in vivo pathology caused by trans-4-hydroxy-2-hexenal appears to be identical to that previously attributed to reactive pyrroles. There are similarities between the toxic effects of this alkenal and those of centrilobular hepatotoxins such as CCl4 and other alkenals formed during lipid peroxidation.

Copper stress induces biosynthesis of octadecanoid and eicosanoid oxygenated derivatives in the brown algal kelp Laminaria digitata.

To better understand the toxicity and the orchestration of antioxidant defenses of marine brown algae in response to copper-induced stress, lipid peroxidation processes were investigated in the brown alga Laminaria digitata. The expression of genes involved in cell protection and anti-oxidant responses were monitored by semi-quantitative reverse transcriptase polymerase chain reaction and the lipid peroxidation products were further characterized by profiling oxylipin signatures using high-pressure liquid chromatography-mass spectrometry. Exposure to copper excess triggers lipoperoxide accumulation and upregulates the expression of stress related genes. It also increases the release of free polyunsaturated fatty acids, leading to an oxidative cascade through at least two distinct mechanisms. Incubations in presence of inhibitors of lipoxygenases and cycloxygenases showed that in addition to the reactive oxygen species-mediated processes, copper stress induces the synthesis of oxylipins through enzymatic mechanisms. Among complex oxylipins, cyclopentenones from C18 and C20 fatty acids such as 12-oxo-PDA and prostaglandins were detected for the first time in brown algae, as well as unique compounds such as the 18-hydroxy-17-oxo-eicosatetraenoic acid. These results suggest that lipid peroxidation participates in the toxic effects of copper and that lipid peroxidation derivatives may regulate protective mechanisms by employing plant-like octadecanoid signals but also eicosanoid oxylipins which are absent in vascular plants.

Lipoxygenase distribution in coffee (Coffea arabica L.) berries.

In this paper lipoxygenase (LOX) presence was investigated in coffee berries to determine its involvement in lipid degradative metabolism of plants grown in organic and conventional cultivations. An immunochemical analysis has evidenced a ca. 80 kDa protein, cross-reacting with an anti-LOX antibody, only in the pulp fraction of berries obtained from plants of both cultivations. LOX activity in this fraction could be monitored either as conjugated diene formation or reaction products (determined by HPLC) and was mainly associated with a heavy membrane fraction (HMF, enriched in tonoplast, endoplasmic reticulum, plasma membrane, and mitochondria) and a light membrane fraction (LMF, enriched in plasma membrane and endoplasmic reticulum, with low levels of tonoplast and mitochondria). The LOX activity of LMF from berries of both cultivations showed an optimum at pH 8.0. The HMF exhibited a different activity peak in samples from conventional (pH 8.0) and organic (pH 5.5) cultures, suggesting the presence of different isoenzymes. These findings were also confirmed by variation of the ratio of 9- and 13-hydroperoxides in organic (1:1) and conventional cultivations (1:10), indicating that the organic one was subjected to an oxidative stress in the coffee pulp fraction leading to the expression of an acidic LOX. Such de novo synthesized LOX activity could be responsible for the production of secondary metabolites, which may interfere with the organoleptic profile of coffee.

Dietary docosahexaenoic acid-induced generation of liver lipid peroxides is not suppressed further by elevated levels of glutathione in ODS rats.

OBJECTIVES: We examined the effects of ascorbic acid (AsA) and glutathione (GSH; experiment 1) and of GSH in acetaminophen-fed rats (experiment 2) on dietary docosahexaenoic acid (DHA)-induced tissue lipid peroxidation. METHODS: In experiment 1, AsA-requiring Osteogenic Disorder Shionogi/Shi-od/od (ODS) rats were fed soybean protein diets containing DHA (10.0% total energy) and AsA at 50 (low) or 300 (normal) mg/kg without (low) or with (normal) methionine at 2 g/kg for 32 d. In experiment 2, ODS rats were fed diets containing DHA (7.8% total energy) and acetaminophen (4 g/kg) with different levels of dietary methionine (low, moderate, high, and excessive at 0, 3, 6, and 9 g/kg, respectively) for 30 d. Tissue lipid peroxides and antioxidant levels were determined. RESULTS: In experiment 1, liver lipid peroxide levels in the low-AsA group were lower than those in the normal-AsA group, but kidney and testis lipid peroxide levels in the low-AsA group were higher than those in the normal-AsA group. Dietary methionine tended to decrease tissue lipid peroxide levels but did not decrease vitamin E (VE) consumption. In experiment 2, a high level of methionine (6 g/kg) decreased liver lipid peroxide levels and VE consumption. However, generation of tissue lipid peroxides and VE consumption were not decreased further by a higher dose of methionine (9 g/kg). CONCLUSIONS: Higher than normal levels of dietary methionine are not necessarily associated with decreased dietary DHA-induced generation of tissue lipid peroxides and VE consumption except that the GSH requirement is increased in a condition such as acetaminophen feeding.

Antioxidative activity and ameliorative effects of memory impairment of sulfur-containing compounds in Allium species.

The antioxidative activity and ameliorative effects on memory impairment by sulfur-containing compounds which occur in Allium vegetables such as onion and garlic were investigated. The antioxidative activities of S-alk(en)yl-L-cysteines and their sulfoxides, volatile alk(en)yl disulfides and trisulfides, and vinyldithiins were examined by using human low-density lipoprotein. It was elucidated that the alk(en)yl substituents and the number of sulfur atoms in the compounds were important for the antioxidative activities. To demonstrate the ameliorative effects on memory impairment, onion extract and synthesized di-n-propyl trisulfide were administered to senescence-accelerated mouse P8. The behavioral experiments showed that onion extract and di-n-propyl trisulfide had highly ameliorative effect of memory impairment. Furthermore, it was found that the hippocampus lipid hydroperoxide in senescence-accelerated mouse P8 was decreased by the administration of di-n-propyl trisulfide. These results suggest that di-n-propyl trisulfide contained in onion ameliorates memory impairment in SAMP8 mouse by its antioxidant effect.

Impact of phyto-oxylipins in plant defense.

Phyto-oxylipins are metabolites produced in plants by the oxidative transformation of unsaturated fatty acids via a series of diverging metabolic pathways. Biochemical dissection and genetic approaches have provided compelling evidence that these oxygenated derivatives actively participate in plant defense mechanisms. During the past decade, interest in this field was focused on the biosynthesis of jasmonic acid (one branch of C18 polyunsaturated fatty acid metabolism) and on its relationship to the other plant defense-signaling pathways. However, recently, antisense strategies have revealed that oxylipins other than jasmonates are probably also essential for the resistance of plants to pathogens.

Probable free radical effects on rat liver nuclei during early hepatocarcinogenesis with a choline-devoid low methionine diet.

Fischer-344 rats fed a choline-devoid diet show lipid peroxidation in the liver nuclei, beginning at 1 day, reaching a peak at 3 days, and subsequently declining by 35 days. Lipid peroxidation in the mitochondria was seen first at 3 days, increased to a maximum at 28 days, and decreased after 35 days to undetectable values at 49 days. Lipid peroxidation was found in both nuclear and mitochondrial fractions both before and after stripping of their outer membranes. No microsomal lipid peroxidation could be detected at any time up to 63 days. The animals fed the same diet supplemented with choline showed no lipid peroxidation in any liver fraction. Animals given CCl4 showed the expected lipid peroxidation in the microsomes but not in the nuclear fraction. The administration of the free radical trapping agent, N-tert-butyl-alpha-phenylnitrone, prevented completely or almost so, microsomal lipid peroxidation induced by CCl4 and nuclear lipid peroxidation in the animals fed the choline-devoid, low methionine diet. The genesis of free radicals in the livers of rats fed a choline-devoid diet is considered as a likely hypothesis for the observed lipid peroxidation. The lipid peroxidation in turn is considered to be closely related to the induction of liver cell death and to the production of alterations in DNA. The DNA alterations coupled with regenerative liver cell proliferation suggest an attractive hypothesis for the initiation of hepatocarcinogenesis in rats fed a choline-devoid diet.

The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma.

The Total (Peroxyl) Radical-trapping Antioxidant Parameter (TRAP) of six freshly prepared human plasma samples and 45 frozen plasma samples has been determined. It is shown that contributions from urate (35-65%), plasma proteins (10-50%), ascorbate (0-24%) and vitamin E (5-10%) to TRAP account for all of the peroxyl radical-trapping antioxidant activity in the majority of the samples. The changes in concentrations of the plasma antioxidants during peroxyl radical attack show that the first line of defense is provided by the plasma sulfhydryl groups, even urate being spared during the initial stages of the reaction. The modes of action of all of these plasma antioxidants and possible interactions between them are discussed, with particular emphasis on the abilities of the water-soluble antioxidants to regenerate or spare the only lipid-soluble antioxidant, vitamin E.

Inverse relationship of ethane or n-pentane and malondialdehyde formed during lipid peroxidation in rat liver microsomes with different oxygen concentrations.

When we incubated rat liver microsomes with ferrous ions and an NADPH-regenerating system, ethane and n-pentane formation increased correspondingly with decreasing concentrations of oxygen in the atmosphere above the incubation, whereas malondialdehyde increased with increasing oxygen concentrations up to a plateau. At very low oxygen concentrations - 100% helium as atmosphere, but presumably traces of oxygen were present in the microsomes - ethane and n-pentane formation were maximal and dependent on the concentrations of ferrous ions, in the case of ethane, a peak being reached at about 20 microM Fe2+, whereas n-pentane continuously increased with increasing concentrations of Fe2+. It is suggested that the inverse relationship of ethane or n-pentane and malondialdehyde is due to two different reaction sequences of microsomal lipid peroxidation with different oxygen sensitivities.

Nephrotoxicity and its prevention by vitamin E in ferric nitrilotriacetate-promoted lipid peroxidation.

Iron and aluminum complexes of nitrilotriacetic acid cause severe nephrotoxicity in Wistar rats. In addition, a high incidence of renal cell carcinoma is seen in ferric nitrilotriacetate-treated animals. The present study was performed to see if lipid peroxidation is involved in ferric nitrilotriacetate toxicity. Ferric nitrilotriacetate had more bleomycin-detectable 'free' iron than any ferric salt, while iron complexed with desferrioxamine or ferric chondroitin sulfate had none. The toxicity of ferric nitrilotriacetate in vivo was more pronounced in vitamin E-deficient rats. A thiobarbituric acid-reactive substance was present in the kidneys of vitamin E-deficient rats in amounts markedly elevated compared to vitamin E-sufficient, or vitamin E-supplemented rats. Non-complexed nitrilotriacetate or aluminum nitrilotriacetate did not produce any thiobarbituric acid-reactive substance in vitamin E-sufficient rats died by the 58th day of administration. We suggest that the iron-stimulated production of free radicals leading to lipid peroxidation is the major cause of ferric nitrilotriacetate-mediated renal toxicity. Vitamin E, a known scavenger of free radicals, is effective in protecting against this iron-induced toxicity.

The use of cis-parinaric acid to determine lipid peroxidation in human erythrocyte membranes. Comparison of normal and sickle erythrocyte membranes.

The recently developed parinaric acid assay is shown to offer possibilities for studying peroxidation processes in biological membrane systems. Taking the human erythrocyte membrane as a model, several initiating systems were investigated, as well as the effect of residual hemoglobin in ghost membrane preparations. The effectivity of a radical generating system appeared to be strongly dependent upon whether radicals are generated at the membrane level or in the water phase. Thus, cumene hydroperoxide at concentrations of 1.0-1.5 mM was found to be a very efficient initiator of peroxidation in combination with submicromolar levels of hemin-Fe3+ as membrane-bound cofactor. In combination with cumene hydroperoxide, membrane-bound hemoglobin appeared to be about 6-times more effective in promoting peroxidation than hemoglobin in the water phase. Results comparing the behaviour of normal and sickle erythrocyte ghost suspensions in the peroxidation assay suggest that the increased oxidative stress on sickle erythrocyte membranes could be due to enhanced membrane binding of sickle hemoglobin, but also partly to a characteristically higher capability of sickle hemoglobin to promote peroxidation. The order of peroxidation-promoting capabilities that could be derived from the experiments was hemin greater than sickle hemoglobin greater than normal hemoglobin.

Relationship between mitochondrial lipid peroxidation and alpha-tocopherol levels in the guinea-pig adrenal cortex.

Lipid peroxidation in mitochondria from the functionally distinct inner (zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea-pig adrenal cortex was investigated. Ferrous ion (Fe2+)-induced lipid peroxidation was far greater in inner than outer zone mitochondria. Ascorbic acid similarly initiated lipid peroxidation to a greater extent in inner zone mitochondrial preparations. Differences in the unsaturated fatty acid content of inner and outer zone mitochondria could not account for the regional differences in lipid peroxidation. Total fatty acid concentrations were greater in the outer than in the inner zone, and the relative amounts of each fatty acid were similar in the two zones. However, mitochondrial concentrations of alpha-tocopherol, an antioxidant known to inhibit lipid peroxidation, were approx. 5-times greater in the outer than inner zone. The results demonstrate that there are regional differences in mitochondrial lipid peroxidation in the adrenal cortex which may be attributable to differences in alpha-tocopherol content. Thus, alpha-tocopherol may serve to protect outer zone mitochondrial enzymes from the consequences of lipid peroxidation and thereby contribute to some of the functional differences between the zones of the adrenal cortex.

Superoxide dismutase depletion and lipid peroxidation in rat liver microsomal membranes: correlation with liver carcinogenesis.

The depletion of superoxide dismutase in the liver of rats held on a copper-deficient diet for 8 weeks induces two profound modifications in microsomal membrane characteristics. These membranes show: (1) a low degree of peroxidation induced in vitro by both endogenous (NADPH and tert-butylhydroperoxide) and exogenous sources (xanthine/xanthine oxidase) of oxygen radicals as revealed by malondialdehyde and diene-conjugate production; (2) a strong decrease of polyunsaturated and an increase of monounsaturated fatty acid content. These alterations are similar to those found in microsomal membranes from fast-growing hepatomas which exhibit a pronounced saturation of fatty acid pattern and lack superoxide dismutase. These observations support the hypothesis that during hepatocarcinogenesis the loss of superoxide dismutase causes an oxidative stress that increases cellular membrane lipid peroxidation, as a consequence of which the cell responds by synthesizing more saturated fatty acids that permanently modify cell membrane structure and properties.

Interaction of lipid peroxidation products with nuclear macromolecules.

The interaction of lipid peroxidation products with nuclear macromolecules was investigated in rat liver nuclei labelled with [3H]arachidonic acid. Lipid peroxidation reactions were driven both non-enzymatically and enzymatically by the addition of ascorbate-Fe2+ or NADPH-ADP-Fe3+, respectively, to the incubation mixtures. The extent of peroxidation was evaluated by the formation of thiobarbituric acid chromophore and of radioactive hydrophilic peroxidation products. The results obtained show that: (1) nuclear membrane lipid peroxidation products formed during incubation interact with DNA and total nuclear proteins; (2) non-enzymatic lipid peroxidation processes induced a 40% larger association of peroxidation products to DNA compared to processes driven enzymatically, whereas the corresponding interaction with total nuclear proteins was similar in both peroxidation systems; (3) the radioactivity associated with histones decreased during incubation in the presence of ascorbate-Fe2+ or NADPH-ADP-Fe3+, and increased in control samples (no additions); (4) inhibition of lipid peroxidation by the iron chelator Desferrioxamine B prevented the association of peroxidation products to nuclear macromolecules; (5) the levels of radioactivity found in DNA after 180 min of incubation would represent the formation of 0.6-1.0 adducts per 10(6) DNA bases. The results obtained provide evidence for an interaction between lipid peroxidation products and chromatin in the interior of the cell nucleus.

Glutathione-dependent inhibition of lipid peroxidation by a soluble, heat-labile factor in animal tissues.

A glutathione-dependent, cytosolic factor (previously thought to be glutathione peroxidase), inhibits lipid peroxidation in both microsomal and mitochondrial membranes. Studies in this laboratory had shown that the inhibition was due to prevention of peroxidative attack on the polyunsaturated fatty acids in the membrane lipids even under conditions that would otherwise promote rapid lipid peroxidation. A glutathione-dependent factor is also present in rat liver cytosol which can utilize peroxides of both free fatty acid salts in solution and free fatty acids in micellar suspension as substrates. It does not, however, utilize peroxidized lipids of microsomal and mitochondrial membranes as substrates. Whether or not this is the same factor which inhibits lipid peroxidation is not known with certainty, but current information indicates that they are not the same. Data presented in this report support the conclusion that neither glutathione peroxidase nor glutathione S-transferase activities appear to be responsible for the inhibition of lipid peroxidation in biological membranes. After partial purification of active preparations of both of these peroxidases, it was observed that neither preparation inhibited lipid peroxidation. The results of this study further support the conclusion that the glutathione-dependent cytosolic factor which inhibits lipid peroxidation in biological membranes does so by preventing the peroxidation rather than by reducing lipid peroxides.

Lipid peroxidation in hypertrophic rat kidney.

During compensatory growth of kidney, microsomal lipid peroxidation is unchanged in the hypertrophy phase and is doubled in a period of hyperplasia. The maximum lipid peroxidation is preceded by a 2-fold increase in the content of cytochrome P-450. Both in microsomes and cytosol, intense peroxidation of lipids is accompanied by a decrease in glutathione content.