Cholesterol Hydroperoxides Research Articles

Abstract PS12-04: Inhibition of GPX4 enhances CDK4/6 inhibitor activity in breast cancer

Abstract Background: Cyclin D-dependent kinases 4 and 6 (CDK4/6) inhibitors (CDK4/6i) including palbociclib, ribociclib and abemaciclib in combination with endocrine therapy are the standard of care for patients with estrogen receptor-positive (ER+). Despite the success of these treatments, cytostasis is frequently observed, and novel strategies that enhance efficacy are required to eradicate residual cancer in the clinic. Methods: In the search of an effective drug combinations to enhance the efficacy of CDK4/6 inhibitors in breast cancer, we performed a whole genome CRISPR-Cas9 suppressor screen in MCF-7, an ER+ model. We also carried out transcriptomics, proteomics, and functional molecular biology analysis of breast cancer models treated with palbociclib to identify resistant mechanisms. Results: The whole genome CRISPR-Cas9 screen revealed that multiple genes involved in oxidative stress and ferroptosis modulated palbociclib sensitivity, being GPX4 the top sensitizing hit. GPX4 is a key glutathione peroxidase that protects again ferroptosis by catalysing the reduction of phospholipid and cholesterol hydroperoxides. CRISPR-depletion or drug inhibition of GPX4 increased sensitivity to palbociclib in ER+ breast cancer models, and in addition sensitised triple negative breast cancer models to palbociclib. Moreover, GPX4 null xenografts were highly sensitive to palbociclib in vivo. Transcriptomics and proteomics analysis showed that palbociclib upregulate pathways involved in oxidative stress and lipid metabolism promoting a redox-lipid imbalance. Palbociclib induced lipid peroxidation leading to a ferroptosis vulnerable state with GPX4 preventing cell death. Polyunsaturated fatty acids (PUFAs) are highly susceptible of lipid peroxidation, and pathways that regulate their abundance in membrane phospholipids were explored. Interestedly, in ER+ breast cancer models, lipid peroxidation relied on a peroxisome AGPAT3-dependent pathway rather than the classical ACSL4 pathway. Conclusion: Our studies demonstrate that quiescence induced by palbociclib results in a ferroptosis-vulnerable state that could be exploited through combination with GPX4 inhibitors to enhance sensitivity to CDK4/6 inhibition in breast cancer. Citation Format: Maria Teresa Herrera Abreu, Uzma Khalid, Jian Ning, Rosalind Cutts, Gemma Wilson, Christopher Lord, Amanda Swain, Nicholas Turner. Inhibition of GPX4 enhances CDK4/6 inhibitor activity in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS12-04.

Protection of Membrane Contact Protein by the Methionine Sulfoxide Reductases.

In this News and Views, I discuss our recent publication that established how steroidogenic acute regulatory-related lipid transfer domain-3 (STARD3), a membrane contact protein situated at lysosomal membranes, plays a role in the detoxification of cholesterol hydroperoxide. STARD3's methionine residues can be oxidized to methionine sulfoxide by cholesterol hydroperoxide, after which methionine sulfoxide reductases reduce the methionine sulfoxide residues back to methionine. The reaction also results in the reduction of the cholesterol hydroperoxide to an alcohol. The cyclic oxidation and reduction of methionine residues in STARD3 at membrane contact sites creates a catalytically efficient mechanism for detoxification of cholesterol hydroperoxide during cholesterol transport, thus protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Cholesterol is more readily oxidized than phospholipid linoleates in cell membranes to produce cholesterol hydroperoxides

Cholesterol is an essential component of cell membranes and serves as an important precursor of steroidal hormones and bile acids, but elevated levels of cholesterol and its oxidation products have been accepted as a risk factor for maintenance of health. The free and ester forms of cholesterol and fatty acids are the two major biological lipids. The aim of this hypothesis paper is to address the long-standing dogma that cholesterol is less susceptible to free radical peroxidation than polyunsaturated fatty acids (PUFAs). It has been observed that cholesterol is peroxidized much slower than PUFAs in plasma but that, contrary to expectations from chemical reactivity toward peroxyl radicals, cholesterol appears to be more readily autoxidized than linoleates in cell membranes. The levels of oxidation products of cholesterol and linoleates observed in humans support this notion. It is speculated that this discrepancy is ascribed to the fact that cholesterol and phospholipids bearing PUFAs are localized apart in raft and non-raft domains of cell membranes respectively and that the antioxidant vitamin E distributed predominantly in the non-raft domains cannot suppress the oxidation of cholesterol lying in raft domains which are relatively deficient in antioxidant.

Cholesterol Hydroperoxide Co-trafficking in Testosterone-generating Leydig Cells: GPx4 Inhibition of Cytotoxic and Anti-steroidogenic Effects

Trafficking of intracellular cholesterol (Ch) to and into mitochondria of steroidogenic cells is required for steroid hormone biosynthesis. This trafficking is typically mediated by one or more proteins of the steroidogenic acute regulatory (StAR) family. Our previous studies revealed that 7-OOH, a redox-active cholesterol hydroperoxide, could be co-trafficked with Ch to/into mitochondria of MA-10 Leydig cells, thereby inducing membrane lipid peroxidation (LPO) which impaired progesterone biosynthesis. These negative effects of 7-OOH were inhibited by endogenous selenoperoxidase GPx4, indicating that this enzyme could protect against 7-OOH-induced oxidative damage/dysfunction. In the present study, we advanced our Leydig focus to cultured murine TM3 cells and then to primary cells from rat testis, both of which produce testosterone. Using a fluorescent probe, we found that extensive free radical-mediated LPO occurred in mitochondria of stimulated primary Leydig cells during treatment with liposomal Ch+7-OOH, resulting in a significant decline in testosterone output relative to that with Ch alone. Strong enhancement of LPO and testosterone shortfall by RSL3 (a GPx4 inhibitor) and reversal thereof by Ebselen (a GPx4 mimetic), suggested that endogenous GPx4 was playing a key antioxidant role. 7-OOH in increasing doses was also cytotoxic to these cells, RSL3 exacerbating this in Ebselen-reversable fashion. Moreover, GPx4 knockdown increased cell sensitivity to LPO with reduced testosterone output. These findings, particularly with primary Leydigs (which best represent cells in intact testis) suggest that GPx4 plays a key protective role against peroxidative damage/dysfunction induced by 7-OOH co-trafficking with Ch.

Methionine sulfoxide reductases and cholesterol transporter STARD3 constitute an efficient system for detoxification of cholesterol hydroperoxides

Methionine sulfoxide reductases (MSRs) are key enzymes in the cellular oxidative defense system. Reactive oxygen species oxidize methionine residues to methionine sulfoxide, and the methionine sulfoxide reductases catalyze their reduction back to methionine. We previously identified the cholesterol transport protein STARD3 as an invivo binding partner of MSRA (methionine sulfoxide reductase A), an enzyme that reduces methionine-S-sulfoxide back to methionine. We hypothesized that STARD3 would also bind the cytotoxic cholesterol hydroperoxides and that its two methionine residues, Met307 and Met427, could be oxidized, thus detoxifying cholesterol hydroperoxide. We now show that in addition to binding MSRA, STARD3 binds all three MSRB (methionine sulfoxide reductase B), enzymes that reduce methionine-R-sulfoxide back to methionine. Using pure 5, 6, and 7 positional isomers of cholesterol hydroperoxide, we found that both Met307 and Met427 on STARD3 are oxidized by 6α-hydroperoxy-3β-hydroxycholest-4-ene (cholesterol-6α-hydroperoxide) and 7α-hydroperoxy-3β-hydroxycholest-5-ene (cholesterol-7α-hydroperoxide). MSRs reduce the methionine sulfoxide back to methionine, restoring the ability of STARD3 to bind cholesterol. Thus, the cyclic oxidation and reduction of methionine residues in STARD3 provides a catalytically efficient mechanism to detoxify cholesterol hydroperoxide during cholesterol transport, protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Anti-steroidogenic effects of cholesterol hydroperoxide trafficking in MA-10 Leydig cells: Role of mitochondrial lipid peroxidation and inhibition thereof by selenoperoxidase GPx4

Steroid hormone synthesis in steroidogenic cells requires cholesterol (Ch) delivery to/into mitochondria via StAR family trafficking proteins. In previous work, we discovered that 7-OOH, an oxidative stress-induced cholesterol hydroperoxide, can be co-trafficked with Ch, thereby causing mitochondrial redox damage/dysfunction. We now report that exposing MA-10 Leydig cells to Ch/7-OOH-containing liposomes (SUVs) results in (i) a progressive increase in fluorescence probe-detected lipid peroxidation in mitochondrial membranes, (ii) a reciprocal decrease in immunoassay-detected progesterone generation, and ultimately (iii) loss of cell viability with increasing 7-OOH concentration. No significant effects were observed with a phospholipid hydroperoxide over the same concentration range. Glutathione peroxidase GPx4, which can catalyze lipid hydroperoxide detoxification, was detected in mitochondria of MA-10 cells. Mitochondrial lipid peroxidation and progesterone shortfall were exacerbated when MA-10 cells were treated with Ch/7-OOH in the presence of RSL3, a GPx4 inhibitor. However, Ebselen, a selenoperoxidase mimetic, substantially reduced RSL3's negative effects, thereby partially rescuing the cells from peroxidative damage. These findings demonstrate that co-trafficking of oxidative stress-induced 7-OOH can disable steroidogenesis, and that GPx4 can significantly protect against this.

Intermembrane Translocation of Photodynamically Generated Lipid Hydroperoxides: Broadcasting of Redox Damage.

Lipid hydroperoxides (LOOHs), including cholesterol- and phospholipid-derived species, are reactive intermediates that arise during photosensitized peroxidation of unsaturated lipids in biological membranes. These intermediates may appear in cancer cell membranes during anti-tumor photodynamic therapy (PDT). Photodynamically generated LOOHs have several different fates, including (a) iron-catalyzed one-electron reduction to free radical species which trigger damaging chain peroxidation reactions, (b) selenoperoxidase-catalyzed two-electron reduction to redox-inert alcohols (LOHs), and (c) spontaneous or protein-mediated translocation to other lipid membrane compartments where (a) or (b) may take place. These different LOOH fates will be described in this review, but with special attention to category (c), which the authors were the first to describe and characterize. Seminal early findings on cholesterol hydroperoxide (ChOOH) translocation and its potential negative consequences will be discussed. In reviewing this work, we wish to congratulate Jean Cadet, for his many outstanding accomplishments as a photobiologist and P&P editor.

Lysine Reacts with Cholesterol Hydroperoxide to Form Secosterol Aldehydes and Lysine-Secosterol Aldehyde Adducts

Two cholesterol secosterol aldehydes, namely, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxyaldehyde (secosterol B), are highly bioactive compounds which have been detected in human tissues and potentially contribute to the development of physiological dysfunctions such as atherosclerosis, Alzheimer’s disease, diabetes, and cancer. They were originally considered to be exclusive products of cholesterol ozonolysis and thus to be evidence for endogenous ozone formation. However, it was recently postulated that primary amines such as lysine may catalyse their formation from cholesterol-5α-hydroperoxide (Ch-5α-OOH), the main product of the oxidation of cholesterol with singlet oxygen. This involves cyclization of Ch-5α-OOH to an unstable dioxetane intermediate, which decomposes to form secosterol aldehydes with triplet carbonyl groups, whose return to the singlet state is at least partly coupled to the conversion of triplet molecular oxygen to singlet oxygen. Here, we subjected cholesterol to photosensitized oxidation, which predominantly produces Ch-5α-OOH and minor amounts of the 6α- and 6β-hydroperoxides, exposed the hydroperoxide mixture to lysine in the presence of the antioxidant 2,6-ditertiary-butyl-4-hydroxytoluene (BHT), and analysed the reaction mixture by liquid chromatography-electrospray ionization-mass spectrometry. Consistent with the postulated lysine-catalysed formation of secosterol aldehydes, we detected formation of the latter and several types of their lysine adducts, including carbinolamines, Schiff’s bases, and amide-type adducts. We propose that the amide type adducts, which are major biomarkers of lipid oxidation, are mainly formed by singlet oxygen-mediated oxidation of the carbinolamine adducts.

H-Atom Abstraction vs Addition: Accounting for the Diverse Product Distribution in the Autoxidation of Cholesterol and Its Esters.

We recently communicated that the free-radical-mediated oxidation (autoxidation) of cholesterol yields a more complex mixture of hydroperoxide products than previously appreciated. In addition to the epimers of the major product, cholesterol 7-hydroperoxide, the epimers of each of the regioisomeric 4- and 6-hydroperoxides are formed as is the 5α-hydroperoxide in the presence of a good H-atom donor. Herein, we complete the story by reporting the products resulting from competing peroxyl radical addition to cholesterol, the stereoisomeric cholesterol-5,6-epoxides, which account for 12% of the oxidation products, as well as electrophilic dehydration products of the cholesterol hydroperoxides, 4-, 6-, and 7-ketocholesterol. Moreover, we interrogate how their distribution-and abundance relative to the H-atom abstraction products-changes in the presence of good H-atom donors, which has serious implications for how these oxysterols are used as biomarkers. The resolution and quantification of all autoxidation products by LC-MS/MS was greatly enabled by the synthesis of a new isotopically labeled cholesterol standard and corresponding selected autoxidation products. The autoxidation of cholesteryl acetate was also investigated as a model for the cholesterol esters which abound in vivo. Although esterification of cholesterol imparts measurable stereoelectronic effects, most importantly reflected in the fact that it autoxidizes at 4 times the rate of unesterified cholesterol, the product distribution is largely similar to that of cholesterol. Deuteration of the allylic positions in cholesterol suppresses autoxidation by H-atom transfer (HAT) in favor of addition, such that the epoxides are the major products. The corresponding kinetic isotope effect ( kH/ kD ∼ 20) indicates that tunneling underlies the preference for the HAT pathway.

26 - Trafficking of cholesterol hydroperoxides to macrophage mitochondria: implications for atherogenesis under oxidative stress

Under oxidative stress conditions associated with chronic inflammation and obesity, scavenger receptor-expressing macrophages in arterial subendothelial spaces can internalize oxidized low-density lipoprotein (oxLDL) in unregulated fashion, potentially giving rise to foam cell-containing atherosclerotic lesions (plaques). In addition to other oxidized lipids, oxLDL contains cholesterol oxides such as 7-hydroperoxide (7-OOH), 7-alcohol (7-OH), and 7-ketone (7=O), all of which can be detected in atherosclerotic plaques. 7-OOH being highly reactive, generates 7-OH and 7=O via free radical redox turnover, so 7-OH/7=O levels typically far exceed those of 7-OOH. To limit adverse buildup of cholesterol and its oxides, macrophages export these lipids to HDL and other acceptors via reverse cholesterol transport (RCT). Several intracellular proteins are involved in RCT, including StarD1, which transports cholesterol into mitochondria (Mito) for conversion to 27-hydroxycholesterol (27-OH) by Cyp27A1 hydroxylase, 27-OH then signaling for ABCA1/G1 transporter expression and cholesterol efflux. Although effects of redox-inactive 7=O/7-OH have been widely studied, little attention has been directed to potentially more dangerous 7-OOH, which can be StarD1-delivered into Mito along with cholesterol, as we recently discovered. We also observed that 7-OOH can stimulate Mito replication (mitogenesis). The significance of 7-OOH trafficking with regard to cardiovascular disease will be discussed. (Supported by NCN grant 2017/26/M/NZ3/01232)

Secondary Dark Reactions Following Photodynamic Treatment are More Damaging Than Previously Thought.

Photodynamic treatment is often thought to produce reactive oxygen species (ROS) that directly induce killing; the nomenclature and phrases revolve around such notions of light-dependency. Few studies reference the possible existence of oxidation products formed in secondary reactions, which bear cytotoxicity competitive to their ROS precursors. Here, we highlight the paper by Girotti and Korytowski in this issue of Photochemistry and Photobiology, which does just that. In this paper, they report on cholesterol hydroperoxides, which are formed after photosensitized oxidation and yield cytotoxic mixtures in dark reactions after the light's turned off. Some of the hydroperoxides are transported by protein carriers and damage tissue outside their site of origin. A similar dark cytotoxicity may be anticipated for biological peroxides from invivo photodynamic therapy.

P-364 - Cholesterol hydroperoxide trafficking to macrophage mitochondria and its implications for atherogenesis under oxidative stress

Under oxidative stress (chronic inflammation, obesity), scavenger receptor-expressing macrophages in arterial subendothelial space internalize oxidized LDL (oxLDL) in unregulated fashion, potentially giving rise to foam cells. OxLDL contains cholesterol oxides such as 7-peroxide (7-OOH), alcohol (7-OH), and ketone (7=O). 7-OOH generates 7-OH and 7=O via free radical-mediated turnover, so 7-OH/7=O levels far exceed those of 7-OOH. To limit adverse buildup of cholesterol and its oxides, macrophages export these lipids to HDL and other acceptors via reverse cholesterol transport (RCT). StarD1 on the outer mitochondrial (Mito) membrane participates in cholesterol transport into Mito for conversion to 27-hydroxycholesterol (27-OH) by Cyp27A1 hydroxylase. The 27-OH then signals for plasma membrane ABCA1/G1 transporter expression and cholesterol efflux. Up to now little attention has been directed to potentially more dangerous 7-OOH (vs. 7-OH and 7=O). Using human THP-1 macrophages, we found that 7-OOH could be StarD1-delivered into Mito along with cholesterol. Upon delivery, 7-OOH triggered free radical-mediated lipid peroxidation, which depolarized the Mito membrane and damaged Cyp27A1, reducing 27-OH output and impairing RCT.

Montmorency tart cherry anthocyanins: dose‐dependent antioxidant activity against cholesterol oxidation

Cardiovascular disease (CVD) is a leading cause of death world‐wide, and cholesterol oxidation (CO) and cholesterol oxidation products (COPs) are widely thought to contribute to CVD by exacerbating mechanisms that produce atherosclerotic plaques. COPs not only hold atherogenic properties, but also mutagenic, carcinogenic and inflammatory properties. More recently, it has been reported that they also play an important role in the onset and progression of neurodegenerative diseases. As promoters of inflammation, cytotoxicity, and fibrogenesis, COPs drive a cycle of inflammation that increases the steady‐state level of radical and non‐radical reactive oxygen species (ROS), which in turn amplify inflammation.Strong evidence indicates that natural products derived from plants, such as anthocyanins, poses antioxidant properties and may inhibit lipid oxidation and its metabolites. Montmorency cherries (Prunus cerasus) contain polyphenols in amounts of 3000 mg/100g dry weight, because of this they could act as a natural source of strong antioxidants. Our group has fractionated and characterized different polyphenols from Montmorency tart cherry extracts. Our preliminary studies indicate that anthocyanins have potent dose‐dependent antioxidants activity against CO suggesting that they may also be efficient at reducing CO‐induced inflammation in the CVD. We explore the antioxidant properties of Montmorency tart cherry anthocyanins against CO using in vitro systems of CVD, such as biomimetic liposomes and low‐density lipoprotein (LDL). Radical‐induced oxidation was triggered by addition of AAPH radical generator at 0.5–2 mM to a large unilamellar vesicles (LUVs) suspension under physiological conditions. Different concentrations of anthocyanins (2–100 μM) were tested over 180 minutes.Results demonstrated that anthocyanins may have a dose‐dependent antioxidant activity, specifically targeting CO. UV‐Vis spectroscopy showed that an increase in the oxidation of liposomal cholesterol when AAPH was added to the system (Control). A dramatic increase in cholesterol oxidation was observed between 180 and 240 minutes. The kinetics associated with the formation of cholesterol hydroperoxides were delayed up 180 min, with subsequent decrease on the accumulation of 7‐ketocholesterol (7‐Keto) and 25‐hydroxycholesterol (25‐OH), which are biomarkers of CO and thus the cardiovascular inflammation. We speculate that specific compounds derived from Montmorency tart cherries have the ability of to penetrate lipid membrane and scavenge ROS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cholesterol Hydroperoxide Generation, Translocation, and Reductive Turnover in Biological Systems.

Cholesterol is like other unsaturated lipids in being susceptible to peroxidative degradation upon exposure to strong oxidants like hydroxyl radical or peroxynitrite generated under conditions of oxidative stress. In the eukaryotic cell plasma membrane, where most of the cellular cholesterol resides, peroxidation leads to membrane structural and functional damage from which pathological states may arise. In low density lipoprotein, cholesterol and phospholipid peroxidation have long been associated with atherogenesis. Among the many intermediates/products of cholesterol oxidation, hydroperoxide species (ChOOHs) have a number of different fates and deserve special attention. These fates include (a) damage-enhancement via iron-catalyzed one-electron reduction, (b) damage containment via two-electron reduction, and (c) inter-membrane, inter-lipoprotein, and membrane-lipoprotein translocation, which allows dissemination of one-electron damage or off-site suppression thereof depending on antioxidant location and capacity. In addition, ChOOHs can serve as reliable and conveniently detected mechanistic reporters of free radical-mediated reactions vs. non-radical (e.g., singlet oxygen)-mediated reactions. Iron-stimulated peroxidation of cholesterol and other lipids underlies a newly discovered form of regulated cell death called ferroptosis. These and other deleterious consequences of radical-mediated lipid peroxidation will be discussed in this review.

Cholesterol Hydroperoxide Trafficking: Impairment of Macrophage Cholesterol Efflux with Implications for Atherogenesis under Oxidative Stress

This study describes a type of “stealthy” delivery of cholesterol 7α/β hydroperoxides (7‐OOH) via a natural cholesterol trafficking pathway. In vascular macrophages this pathway normally delivers cholesterol to mitochondria to initiate export signaling when its level becomes excessive. Because 7‐OOHs are structurally similar to cholesterol, they can be recognized by steroidogenic acute regulatory (StAR) proteins and trafficked to mitochondria alongside cholesterol.Oxidative stress associated with cardiovascular disease can produce various oxidized lipids, including cholesterol oxides such as 7‐OOH, 7‐hydroxide (7‐OH), and 7‐ketone (7=O). Unlike 7=O and 7‐OH, 7‐OOH is redox‐active, giving rise to the others via potentially toxic free radical reactions. In the present study we tested the novel hypothesis that under oxidative stress conditions, StAR family proteins not only deliver cholesterol to/into mitochondria of vascular macrophages, but also 7‐OOH, which induces peroxidative damage that impairs early stage reverse cholesterol transport. Stimulation of human monocyte‐derived THP‐1 macrophages with dibutyryl‐cAMP resulted in substantial upregulation of StarD1 and plasma membrane ABCA1 which exports cholesterol to acceptors such as ApoA1. siRNA‐induced StarD1 knockdown (kd) prior to stimulation had no effect on StarD4, but reduced ABCA1 upregulation, linking the latter to StarD1 functionality. Mitochondria in stimulated StarD1‐kd cells internalized 7‐OOH slower than non‐stimulated controls and underwent less 7‐OOH‐induced lipid peroxidation and membrane depolarization, as probed with C11‐BODIPY and JC‐1, respectively. Major functional consequences of 7‐OOH exposure were (i) loss of mitochondrial CYP27A1 activity, (ii) reduced 27‐hydroxycholesterol (27‐OH) output, and (iii) down‐regulation of ABCA1 and ABCG1. Consistently, 7‐OOH‐challenged macrophages exported less cholesterol to apoA‐I or HDL than did non‐challenged controls. StarD1‐mediated 7‐OOH transport was also found to be highly cytotoxic, whereas 7=O and 7‐OH were minimally toxic. These findings support our hypothesis that 7‐OOH found in oxidized low density lipoprotein gets delivered to macrophage mitochondria and promotes atherogenesis through impairment of cholesterol homeostasis.Support or Funding InformationThis work was supported by Polish National Science Center grant 2014/13/B/NZ3/00833 (WK) and by National Institutes of Health grants HL85677 (AWG) and HL58012 (DS). Support of American Heart Association Fellowship 14PRE185800221 (ACC) is also acknowledged

Interaction of plasmenylcholine with free radicals in selected model systems

Plasmalogens (Plg) – naturally occurring glycerophospholipids with the vinyl-ether group in the sn-1 position are generally viewed as physiological antioxidants. Although there are numerous examples of antioxidant action of plasmalogen in cell cultures and in experimental animals, this hypothesis is far from being satisfactorily proven due to substantial limitations of such studies. Thus, plasmalogen reactivity in cells results in the accumulation of toxic byproducts and the experimental design is usually too complicated to evaluate the protective function of solely one type of lipid molecular species. In this study, experiments were performed in homogenous and heterogeneous model systems consisting of solutions in organic solvents as well as micelles and liposomes containing pure synthetic plasmenylcholines. Under the experimental conditions used, chemical reactivity of plasmalogens could be attributed to specific fatty acid esterification pattern. This is important because the chemical reactivity cannot be separated from physico-chemical properties of the lipids. Time-dependent formation of phospholipid and cholesterol hydroperoxides were determined by iodometric assay and HPLC-EC. EPR oximetry and Clark electrode were employed to detect the accompanying changes in oxygen concentration. Oxidation of the studied lipids was monitored by standard colorimetric TBARS method as well as MALDI-TOF mass spectrometry. Our data indicate that the reactivity of sn-2 monounsaturated vinyl ether lipids in peroxyl radical-induced or iron-catalyzed peroxidation reactions is comparable with that of their diacyl analogs. In samples containing cholesterol and plasmalogens, oxidative processes lead to accumulation of the radical oxidation product of cholesterol. It can be concluded that the antioxidant action of plasmalogens takes place intramolecularly rather than intermolecularly and depends on the degree of unsaturation of esterified fatty acids. Thus, it is questionable if plasmalogens can really be viewed as “endogenous antioxidant”, even though they may exhibit, under special conditions, protective effect.

PrxQ B from Mycobacterium tuberculosis is a monomeric, thioredoxin-dependent and highly efficient fatty acid hydroperoxide reductase

Mycobacterium tuberculosis (M. tuberculosis) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant systems could facilitate novel drug developments. In this work, we characterized the product of the gene Rv1608c from M. tuberculosis, which according to sequence homology had been annotated as a putative peroxiredoxin of the peroxiredoxin Q subfamily (PrxQ B from M. tuberculosis or MtPrxQ B). The protein has been reported to be essential for M. tuberculosis growth in cholesterol-rich medium. We demonstrated the M. tuberculosis thioredoxin B/C-dependent peroxidase activity of MtPrxQ B, which acted as a two-cysteine peroxiredoxin that could function, although less efficiently, using a one-cysteine mechanism. Through steady-state and competition kinetic analysis, we proved that the net forward rate constant of MtPrxQ B reaction was 3 orders of magnitude faster for fatty acid hydroperoxides than for hydrogen peroxide (3×106vs 6×103M−1s−1, respectively), while the rate constant of peroxynitrite reduction was (0.6−1.4) ×106M−1s−1 at pH 7.4. The enzyme lacked activity towards cholesterol hydroperoxides solubilized in sodium deoxycholate. Both thioredoxin B and C rapidly reduced the oxidized form of MtPrxQ B, with rates constants of 0.5×106 and 1×106M−1s−1, respectively. Our data indicated that MtPrxQ B is monomeric in solution both under reduced and oxidized states. In spite of the similar hydrodynamic behavior the reduced and oxidized forms of the protein showed important structural differences that were reflected in the protein circular dichroism spectra.

Inhibition of phagocytic activity of ARPE-19 cells by free radical mediated oxidative stress

Oxidative stress is a main factor responsible for key changes leading to the onset of age-related macular degeneration (ARMD) that occur in the retinal pigment epithelium (RPE), which is involved in phagocytosis of photoreceptor outer segments (POS). In this study, hydrogen peroxide (H2O2), H2O2 and iron ions (Fe) or rose Bengal (RB) in the presence of NADH and Fe were used to model free radical mediated oxidative stress to test if free radicals and singlet oxygen have different efficiency to inhibit phagocytosis of ARPE-19 cells. Free radical mediated oxidative stress was confirmed by HPLC-EC(Hg) measurements of cholesterol hydroperoxides in treated cells. Electron paramagnetic resonance (EPR) spin trapping was employed to detect superoxide anion. Cell survival was analyzed by the MTT assay. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS and non-specific phagocytosis of fluorescent beads were measured by flow cytometry. HPLC analysis of cells photosensitized with RB in the presence of NADH and Fe indicated substantial increase in formation of free radical-dependent 7α/7β-hydroperoxides. EPR spin trapping confirmed the photogeneration of superoxide anion in samples enriched with RB, NADH and Fe. For all three protocols sub-lethal oxidative stress induced significant inhibition of the specific phagocytosis of POS. In contrast, non-specific phagocytosis was inhibited only by H2O2 or H2O2 and Fe treatment. Inhibition of phagocytosis was transient and recoverable by 24 h. These results suggest that free radicals may exert similar to singlet oxygen efficiency in inhibiting phagocytosis of RPE cells, and that the effect depends on the location where initial reactive species are formed.

Molecular simulations of the effects of phospholipid and cholesterol peroxidation on lipid membrane properties

Non-enzymatic lipid peroxidation may change biomembrane structure and function. Here, we employed molecular dynamics simulations to study the effects of either phospholipid or cholesterol peroxidation individually, as well as the combined peroxidation of both components. When lipids were peroxidized, the generated OOH groups migrated to the membrane surface and engaged in H-bonds with each other and the phospholipid carbonyl ester groups. It caused the sn-2 acyl chains of phospholipid hydroperoxides to bend and the whole sterol backbone of cholesterol hydroperoxides to tilt. When phospholipids were kept intact, peroxidation of the sterol backbone led to a partial degradation of its condensing and ordering properties, independently of the position and isomerism of the OOH substitution. However, even in massively peroxidized membranes in which all phospholipids and cholesterol were peroxidized, the condensing and ordering properties of the sterol backbone were still significant. The possible implications for the formation of membrane lateral domains were discussed. Cholesterol peroxyl radicals were also investigated and we found that the OO groups did not migrate to the headgroups region.

Oxidation of Cholesterol and Formation of Cholesterol Hydroperoxides Decreases the Cholesterol Concentration at which Formation of Cholesterol Bilayer Domains and Cholesterol Crystals is Initiated in Phospholipid Bilayers

Recently we demonstrated that formation of cholesterol bilayer domains (CBDs) precedes formation of cholesterol crystals (CHCs) in cholesterol/dimyristoylphosphatidylcholine (Chol/DMPC) membranes (Mainali et. al., J. Phys. Chem. B, 117(2013)8994-9003). In the presented studies, we show that the oxidation of Chol in Chol/DMPC membranes induces formation of CBDs and CHCs at significantly lower Chol concentrations than in membranes without oxidation. To preserve compositional homogeneity throughout the membrane suspension we prepared the multilamellar liposomes with a rapid solvent exchange method. The oxidation of Chol was induced using a photosensitizer, rose Bengal, and the extent of oxidation (formation of Chol hydroperoxides) was measured using iodometric assay. To avoid formation of phospholipid hydroperoxides, which would interfere with our assay, the saturated phospholipid (DMPC) was used to prepare liposomes. Saturation recovery electron paramagnetic resonance with spin-labeled cholesterol analogues (androstane and cholestane spin labels) was used to detect the formation of CBDs and differential scanning calorimetry was used to detect the formation of CHCs. We conclude that formation of Chol hydroperoxides induces the formation of CBDs at Chol concentrations where previously CBDs were not present in non-oxidized membranes as well as the collapse of the already formed CBDs to CHCs, presumably outside the lipid bilayer.