Hyperoxidized Species of Heme Have a Potent Capacity to Induce Autoreactivity of Human IgG Antibodies.

The interaction of some human antibodies with heme results in posttranslational acquisition of binding to various self- and pathogen-derived antigens. The previous studies on this phenomenon were performed with oxidized heme (Fe3+). In the present study, we elucidated the effect of other pathologically relevant species of heme, i.e., species that were formed after contact of heme with oxidizing agents such as hydrogen peroxide, situations in which heme's iron could acquire higher oxidation states. Our data reveal that hyperoxidized species of heme have a superior capacity to heme (Fe3+) in triggering the autoreactivity of human IgG. Mechanistic studies demonstrated that oxidation status of iron was of critical importance for the heme's effect on antibodies. We also demonstrated that hyperoxidized heme species interacted at higher affinities with IgG and that this binding occurred through a different mechanism as compared to heme (Fe3+). Regardless of their profound functional impact on the antigen-binding properties of antibodies, hyperoxidized species of heme did not affect Fc-mediated functions of IgG, such as binding to the neonatal Fc receptor. The obtained data contribute to a better understanding of the pathophysiological mechanism of hemolytic diseases and of the origin of elevated antibody autoreactivity in patients with some hemolytic disorders.

Oxidation of catalytic cysteine of human deubiquitinase BAP1 triggers misfolding and aggregation in addition to functional loss.

Deubiquitinating enzymes (DUBs) form a large protease family involved in a myriad of biological and pathological processes, including ROS sensors. ROS-mediated inhibition of their DUB activities is critical for fine-tuning the stress-activated signaling pathways. Here, we demonstrate that the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) is highly sensitive to moderate oxidative stress. Oxidation of the catalytic C91 significantly destabilizes BAP1-UCH and increases the population of partially unfolded form, which is prone to aggregation. Unlike other DUBs, the oxidation-induced structural and functional loss of BAP1-UCH cannot be fully reversed by reducing agents. The oligomerization of oxidized BAP1-UCH is attributed to inter-molecular disulfide bond formation. Hydrogen-deuterium mass exchange spectrometry (HDX-MS) reveals increased fluctuations of the central ß-sheet upon oxidation. Our findings suggest that oxidation-mediated functional loss and increased aggregation propensity may contribute to oncogenesis associated with BAP1.

The Nature of Resistance of the Coagulation Factor XIII Structure to Hypochlorite-Induced Oxidation.

The damage to blood coagulation factor XIII (FXIII) at different stages of its enzymatic activation under the action of various physiological amounts of hypochlorite ion was studied. The results obtained by HPLC-MS/MS, SDS-PAGE, and colorimetry showed that, during the conversion of FXIII to FXIIIa, the vulnerability of FXIII to hypochlorite-induced oxidation increased. FXIII oxidized with 150 µM hypochlorite completely retained its enzymatic activity inherent to the intact protein, whereas FXIIIa treated with 50 µM hypochlorite showed sharply reduced enzymatic activity. It was shown that a number of methionine and cysteine residues on the catalytic subunit can perform antioxidant function; additionally, the regulatory subunits of FXIII-B contribute to the antioxidant protection of the catalytic center of the FXIII-A subunit, which, together with the tight packing of the tetrameric structure of the FXIII proenzyme, are the three factors that provide high protein resistance to the oxidizing agent.

Elucidation of biosynthetic pathway of a plant hormone abscisic acid in phytopathogenic fungi.

Abscisic acid (ABA) is one of the plant hormones that regulates physiological functions in various organisms, including plants, sponges, and humans. The biosynthetic machinery in plants is firmly established, while that in fungi is still unclear. Here, we elucidated the functions of the four biosynthetic genes, bcABA1-bcABA4, found in Botrytis cinerea by performing biotransformation experiments and in vitro enzymatic reactions with putative biosynthetic intermediates. The first-committed step is the cyclization of farnesyl diphosphate to give α-ionylideneethane catalyzed by a novel sesquiterpene synthase, BcABA3, which exhibits low amino acid sequence identities with sesquiterpene synthases. Subsequently, two cytochrome P450s, BcABA1 and BcABA2, mediate oxidative modifications of the cyclized product to afford 1',4'-trans-dihydroxy-α-ionylideneacetic acid, which undergoes alcohol oxidation to furnish ABA. Our results demonstrated that production of ABA does not depend on the nucleotide sequence of bcABA genes. The present study set the stage to investigate the role of ABA in infections.

Peroxisomes and Cellular Oxidant/Antioxidant Balance: Protein Redox Modifications and Impact on Inter-organelle Communication.

Disturbances in cellular redox balance have been associated with pro-aging mechanisms and increased risk for various chronic disease states. Multiple lines of evidence indicate that peroxisomes are central players in cellular redox metabolism. Nevertheless, the potential role of this organelle as intracellular redox signaling platform has been largely overlooked for a long time. Fortunately, this situation is now changing. This review provides a snapshot of the current progress in the field, with an emphasis on the situation in mammals. We first briefly introduce the basics of redox biology and how reactive oxygen and nitrogen species can drive cellular signaling events. Next, we discuss current evidence linking peroxisome (dys)function to redox signaling, both in health and disease. We also highlight what is currently known about the downstream targets of peroxisome-derived oxidants. In addition, we present an extensive list of proteins that are involved in peroxisome functioning and have been identified as being responsive to oxidative stress in large scale redox proteomics studies. Finally, we address how changes in peroxisomal redox state may impact on functional mechanisms underlying inter-organelle communication. Gaining more insight into these mechanisms is key to our understanding of how peroxisomes are embedded in cellular signaling networks implicated in aging and diseases such as cancer, diabetes, and neurodegenerative disorders.

Oxidation-induced modifications of the catalytic subunits of plasma fibrin-stabilizing factor at the different stages of its activation identified by mass spectrometry.

Plasma fibrin-stabilizing factor (pFXIII) is a heterotetrameric proenzyme composed of two catalytic A subunits (FXIII-A2) and two inhibitory/carrier B subunits (FXIII-B2). The main function of the protein is the formation of cross-links between the polypeptide chains of the fibrin clot. The conversion of pFXIII into the enzymatic form FXIII-A2* is a multistage process. Like many other blood plasma proteins, pFXIII is an oxidant-susceptible target. The influence of distinct sites susceptible to oxidation-mediated modifications on the changes in the structural-functional characteristics of the protein remains fully unexplored. For the first time, a set of the oxidation sites within FXIII-A2 under ozone-induced oxidation of pFXIII at different stages of its activation have been identified by mass spectrometry, and the extent as well as the chemical nature of these modifications have been explored. It was shown that the set of amino acid residues susceptible to oxidative attack and the degree of oxidation of these residues in FXIII-A2 of non-activated pFXIII, pFXIII activated by Ca2+ and fully activated pFXIII treated with thrombin and Ca2+ significantly differ. The obtained data enable one to postulate that in the process of the proenzyme conversion into FXIII-A2*, new earlier-unexposed amino acid residues become available for the oxidizer while some of the initially surface-exhibited residues are buried within the protein globule.

Modification in oxidative processes in muscle tissues exposed to laser- and light-emitting diode radiation.

Exposure of living tissues to high-intensity red or near-infrared light can produce the oxidative stress effects both in the target zone and adjacent ones. The protein oxidative modification (POM) products can be used as reliable and early markers of oxidative stress. The contents of modified proteins in the investigated specimens can be evaluated by the 2,4-dinitrophenylhydrazine assay (the DNPH assay). Low-intensity red light is able to decrease the activity of oxidative processes and the DNPH assay data about the POM products in the biological tissues could show both an oxidative stress level and an efficiency of physical agent protection against the oxidative processes. Two control groups of white rats were irradiated by laser light, the first control group by red light and the second one by near-infrared radiation (NIR).Two experimental groups were consequently treated with laser and red low-level light-emitting diode radiation (LED). One of them was exposed to red laser light + LED and the other to NIR + LED. The fifth group was intact. Each group included ten animals. The effect of laser light was studied by methods of protein oxidative modifications. We measured levels of both induced and spontaneous POM products by the DNPH assay. The dramatic increase in levels of POM products in the control group samples when compared with the intact group data as well as the sharp decrease in the POM products in the experimental groups treated with LED low-level light were statistically significant (p ≤ 0.05). Exposure of skeletal muscles to high-intensity red and near-infrared laser light causes oxidative stress that continues not less than 3 days. The method of measurement of POM product contents by the DNPH assay is a reliable test of an oxidative process rate. Red low-intensity LED radiation can provide rehabilitation of skeletal muscle tissues treated with high-intensity laser light.

Pathophysiology of tobacco smoke exposure: recent insights from comparative and redox proteomics.

First-hand and second-hand tobacco smoke are causally linked to a huge number of deaths and are responsible for a broad spectrum of pathologies such as cancer, cardiovascular, respiratory, and eye diseases as well as adverse effects on female reproductive function. Cigarette smoke is a complex mixture of thousands of different chemical species, which exert their negative effects on macromolecules and biochemical pathways, both directly and indirectly. Many compounds can act as oxidants, pro-inflammatory agents, carcinogens, or a combination of these. The redox behavior of cigarette smoke has many implications for smoke related diseases. Reactive oxygen and nitrogen species (both radicals and non-radicals), reactive carbonyl compounds, and other species may induce oxidative damage in almost all the biological macromolecules, compromising their structure and/or function. Different quantitative and redox proteomic approaches have been applied in vitro and in vivo to evaluate, respectively, changes in protein expression and specific oxidative protein modifications induced by exposure to cigarette smoke and are overviewed in this review. Many gel-based and gel-free proteomic techniques have already been used successfully to obtain clues about smoke effects on different proteins in cell cultures, animal models, and humans. The further implementation with other sensitive screening techniques could be useful to integrate the comprehension of cigarette smoke effects on human health. In particular, the redox proteomic approach may also help identify biomarkers of exposure to tobacco smoke useful for preventing these effects or potentially predictive of the onset and/or progression of smoking-induced diseases as well as potential targets for therapeutic strategies.

Oxidative stress and aging: synergies for age related diseases.

Aging is characterized by a progressive decline in physiological function and underlies several disabilities, including the increased sensitivity of cells and tissues to undergo pathological oxidative stress. In recent years, efforts have been made to better understand the relationship between age and oxidative stress and further develop therapeutic strategies to minimize the impact of both events on age-related diseases. In this work, we review the impact of the oxidant and antioxidant systems during aging and disease development and discuss the crosstalk of oxidative stress and other aging processes, with a focus on studies conducted in elderly populations.

A role of methionines in the functioning of oxidatively modified fibrinogen.

Posttranslational modifications in fibrinogen resulting from induced oxidation or oxidative stress in the organism can have deleterious influence on optimal functioning of fibrinogen, causing a disturbance in assembly and properties of fibrin. The protective mechanism supporting the ability of fibrinogen to function in ROS-generating environment remains completely unexplored. The effects of very low and moderately low HOCl/-OCl concentrations on fibrinogen oxidative modifications, the fibrin network structure as well as the kinetics of both fibrinogen-to-fibrin conversion and fibrin hydrolysis have been explored in the current study. As opposed to 25 Μm, HOCl/-OCl, 10 µM HOCl/-OCl did not affect the functional activity of fibrinogen. It is shown for the first time that a number of Met residues, AαMet476, AαMet517, AαMet584, BßMet367, γMet264, and γMet94, identified in 10 µM HOCl/-OCl fibrinogen by the HPLC-MS/MS method, operate as ROS scavengers, performing an important antioxidant function. In turn, this indicates that the fibrinogen structure is adapted to the detrimental action of ROS. The results obtained in our study provide evidence for a protective mechanism responsible for maintaining the structure and functioning of fibrinogen molecules in the bloodstream under conditions of mild and moderate oxidative stress.

Insight into the protein oxidation impact on the surface properties of myofibrillar proteins from bighead carp.

The objective of this study was to elucidate the influence of oxidative modifications of myofibrillar proteins (MPs) on their surface properties. Oxidative modifications (deamination, formation of disulfide bonds and Schiff bases), particle size, net surface charge, and binding ability of volatiles (2-enthylfuran, 1-octen-3-ol, hexanal, and octanal) of oxidized MPs was measured. Molecular docking of volatiles with actomyosin was performed using Qvina-W program and the specific oxidative modifications (monoxidation and deamination) of MPs were determined using LC-MS/MS. Results showed that oxidation of Cys (forming sulfinic, sulfonic, sulfenic acid, and disulfide bonds), monoxidation of Ala, Lys, Glu, and Asn, and deamination of Lys changed the surface properties of oxidized MPs including enhanced surface hydrophobicity and decreased affinity to volatile compounds and water. Overall, this study gives evidence of how protein oxidation affects the properties of MPs and therefore deteriorates fish meat quality.

Proteome-wide quantitative analysis of redox cysteine availability in the Drosophila melanogaster eye reveals oxidation of phototransduction machinery during blue light exposure and age.

The retina is one of the highest oxygen-consuming tissues because visual transduction and light signaling processes require large amounts of ATP. Thus, because of the high energy demand, oxygen-rich environment, and tissue transparency, the eye is susceptible to excess production of reactive oxygen species (ROS) resulting in oxidative stress. Oxidative stress in the eye is associated with the development and progression of ocular diseases including cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy. ROS can modify and damage cellular proteins, but can also be involved in redox signaling. In particular, the thiol groups of cysteines can undergo reversible or irreversible oxidative post-translational modifications (PTMs). Identifying the redox-sensitive cysteines on a proteome-wide scale provides insight into those proteins that act as redox sensors or become irreversibly damaged upon exposure to oxidative stress. In this study, we profiled the redox proteome of the Drosophila eye under prolonged, high intensity blue light exposure and age using iodoacetamide isobaric label sixplex reagents (iodo-TMT) to identify changes in cysteine availability. Although redox metabolite analysis of the major antioxidant, glutathione, revealed similar ratios of its oxidized and reduced form in aged or light-stressed eyes, we observed different changes in the redox proteome under these conditions. Both conditions resulted in significant oxidation of proteins involved in phototransduction and photoreceptor maintenance but affected distinct targets and cysteine residues. Moreover, redox changes induced by blue light exposure were accompanied by a large reduction in light sensitivity that did not arise from a reduction in the photopigment level, suggesting that the redox-sensitive cysteines we identified in the phototransduction machinery might contribute to light adaptation. Our data provide a comprehensive description of the redox proteome of Drosophila eye tissue under light stress and aging and suggest how redox signaling might contribute to light adaptation in response to acute light stress.

Quantitative analysis of redox proteome reveals oxidation-sensitive protein thiols acting in fundamental processes of developmental hematopoiesis.

Fetal and adult hematopoietic stem and progenitor cells (HSPCs) are characterized by distinct redox homeostasis that may influence their differential cellular behavior in normal and malignant hematopoiesis. In this work, we have applied a quantitative mass spectrometry-based redox proteomic approach to comprehensively describe reversible cysteine modifications in primary mouse fetal and adult HSPCs. We defined the redox state of 4,438 cysteines in fetal and adult HSPCs and demonstrated a higher susceptibility to oxidation of protein thiols in fetal HSPCs. Our data identified ontogenic changes to oxidation state of thiols in proteins with a pronounced role in metabolism and protein homeostasis. Additional redox proteomic analysis identified oxidation changes to thiols acting in mitochondrial respiration as well as protein homeostasis to be triggered during onset of MLL-ENL leukemogenesis in fetal HSPCs. Our data has demonstrated that redox signaling contributes to the regulation of fundamental processes of developmental hematopoiesis and has pinpointed potential targetable redox-sensitive proteins in in utero-initiated MLL-rearranged leukemia.

Redox Proteomics Analysis of Atherosclerotic Aortas: Application of the "OxICAT" Method.

Atherosclerosis development and progression have been linked to vascular reactive oxygen species (ROS). Plaque formation and especially instability, frequently resulting in acute coronary syndromes, have been linked to cell apoptosis and senescence, but also mainly to increased cellular oxidative stress. ROS are characterized by their high chemical reactivity and a resulting short half-life. This high reactivity usually involves reversible and/or irreversible protein modifications and specifically the covalent oxidative modification of cysteine residues. The latter can be used for the identification of protein-chemical footprints, leading to indirect monitoring of ROS. Proteomics and especially liquid chromatography tandem mass spectrometry (LC-MS/MS) approaches have emerged as a powerful tool to identify such protein modifications in biological samples (e.g., body fluids, tissues, cells). Application of a well-established quantitative thiol trapping technique termed OxICAT enables the detection and quantification of oxidative thiol modifications of thousands of proteins in a single experiment. In this chapter, a step-by-step guide for the redox proteomics analysis of atherosclerotic aortas, by utilizing the OxICAT method, as optimized by our group is provided.

Molecular Insight into the Regulation of Vimentin by Cysteine Modifications and Zinc Binding.

The intermediate filament protein vimentin is involved in essential cellular processes, including cell division and stress responses, as well as in the pathophysiology of cancer, pathogen infection, and autoimmunity. The vimentin network undergoes marked reorganizations in response to oxidative stress, in which modifications of vimentin single cysteine residue, Cys328, play an important role, and is modulated by zinc availability. However, the molecular basis for this regulation is not fully understood. Here, we show that Cys328 displays a low pKa, supporting its reactivity, and is readily alkylated and oxidized in vitro. Moreover, combined oxidation and crosslinking assays and molecular dynamics simulations support that zinc ions interact with Cys328 in its thiolate form, whereas Glu329 and Asp331 stabilize zinc coordination. Vimentin oxidation can induce disulfide crosslinking, implying the close proximity of Cys328 from neighboring dimers in certain vimentin conformations, supported by our computational models. Notably, micromolar zinc concentrations prevent Cys328 alkylation, lipoxidation, and disulfide formation. Moreover, zinc selectively protects vimentin from crosslinking using short-spacer cysteine-reactive but not amine-reactive agents. These effects are not mimicked by magnesium, consistent with a lower number of magnesium ions hosted at the cysteine region, according to molecular dynamics simulations. Importantly, the region surrounding Cys328 is involved in interaction with several drugs targeting vimentin and is conserved in type III intermediate filaments, which include glial fibrillary acidic protein and desmin. Altogether, our results identify this region as a hot spot for zinc binding, which modulates Cys328 reactivity. Moreover, they provide a molecular standpoint for vimentin regulation through the interplay between cysteine modifications and zinc availability.

Type III intermediate filaments as targets and effectors of electrophiles and oxidants.

Intermediate filaments (IFs) play key roles in cell mechanics, signaling and homeostasis. Their assembly and dynamics are finely regulated by posttranslational modifications. The type III IFs, vimentin, desmin, peripherin and glial fibrillary acidic protein (GFAP), are targets for diverse modifications by oxidants and electrophiles, for which their conserved cysteine residue emerges as a hot spot. Pathophysiological examples of these modifications include lipoxidation in cell senescence and rheumatoid arthritis, disulfide formation in cataracts and nitrosation in endothelial shear stress, although some oxidative modifications can also be detected under basal conditions. We previously proposed that cysteine residues of vimentin and GFAP act as sensors for oxidative and electrophilic stress, and as hinges influencing filament assembly. Accumulating evidence indicates that the structurally diverse cysteine modifications, either per se or in combination with other posttranslational modifications, elicit specific functional outcomes inducing distinct assemblies or network rearrangements, including filament stabilization, bundling or fragmentation. Cysteine-deficient mutants are protected from these alterations but show compromised cellular performance in network assembly and expansion, organelle positioning and aggresome formation, revealing the importance of this residue. Therefore, the high susceptibility to modification of the conserved cysteine of type III IFs and its cornerstone position in filament architecture sustains their role in redox sensing and integration of cellular responses. This has deep pathophysiological implications and supports the potential of this residue as a drug target.

The Structure of Blood Coagulation Factor XIII Is Adapted to Oxidation.

The blood coagulation factor XIII (FXIII) plays a critical role in supporting coagulation and fibrinolysis due to both the covalent crosslinking of fibrin polymers, rendering them resistant to plasmin lysis, and the crosslinking of fibrin to proteins of the fibrinolytic system. The hypochlorite-mediated oxidation of the blood coagulation factor XIII (FXIII) at the different stages of its enzymatic activation is studied for the first time in this paper. The consolidated results obtained with the aid of MS/MS, electrophoresis, and colorimetry demonstrate that in the process of FXIII's conversion into FXIIIa, the vulnerability of FXIII to hypochlorite-induced oxidation increased as follows: native FXIII < FXIII + Ca2+ << FXIII + Ca2+/thrombin. The modification sites were detected among all the structural regions of the catalytic FXIII-A subunit, except for the activation peptide, and embraced several sushi domains of the FXIII-B subunit. Oxidized amino acid residues belonging to FXIII-A are surface-exposed residues and can perform an antioxidant role. The regulatory FXIII-B subunits additionally contribute to the antioxidant defense of the catalytic center of the FXIII-A subunits. Taken together, the present data along with the data from previous studies demonstrate that the FXIII proenzyme structure is adapted to oxidation.

Photobleaching of pheomelanin increases its phototoxic potential: Physicochemical studies of synthetic pheomelanin subjected to aerobic photolysis.

Although melanin is a photoprotective pigment, its elevated photochemical reactivity could lead to various phototoxic processes. Photoreactivity of synthetic pheomelanin, derived from 5-S-cysteinyldopa (5SCD-M) and its photodegradation products obtained by subjecting the melanin to aerobic irradiation with UV-visible light, was examined employing an array of advanced physicochemical methods. Extensive photolysis of 5SCD-M was accompanied by partial bleaching of the melanin, modification of its paramagnetic properties, and significant increase in the ability to photogenerate singlet oxygen. The changes correlated with a substantial decrease in the melanin content of benzothiazine (BT) units and increase of modified benzothiazole (BZ) units. Synthetically prepared BZ exhibited higher efficiency to photogenerate singlet oxygen than the synthetic BT, and the free radical form of BZ, unlike that of BT, did not show measurable spin density on nitrogen atom, which was confirmed by quantum chemical calculations. Formation of modified BZ units in the photobleached 5SCD-M is responsible for the paramagnetic and photochemical changes of the melanin and its elevated phototoxic potential. Given a relatively constant pheomelanin-eumelanin ratio, such undesirable changes could occur in individual of all skin types.

The effect of reactive oxygen and nitrogen species on the structure of cytoglobin: A potential tumor suppressor.

Many current anti-cancer therapies rely on increasing the intracellular reactive oxygen and nitrogen species (RONS) contents with the aim to induce irreparable damage, which subsequently results in tumor cell death. A novel tool in cancer therapy is the use of cold atmospheric plasma (CAP), which has been found to be very effective in the treatment of many different cancer cell types in vitro as well as in vivo, mainly through the vast generation of RONS. One of the key determinants of the cell's fate will be the interaction of RONS, generated by CAP, with important proteins, i.e. redox-regulatory proteins. One such protein is cytoglobin (CYGB), a recently discovered globin proposed to be involved in the protection of the cell against oxidative stress. In this study, the effect of plasma-produced RONS on CYGB was investigated through the treatment of CYGB with CAP for different treatment times. Spectroscopic analysis of CYGB showed that although chemical modifications occur, its secondary structure remains intact. Mass spectrometry experiments identified these modifications as oxidations of mainly sulfur-containing and aromatic amino acids. With longer treatment time, the treatment was also found to induce nitration of the heme. Furthermore, the two surface-exposed cysteine residues of CYGB were oxidized upon treatment, leading to the formation of intermolecular disulfide bridges, and potentially also intramolecular disulfide bridges. In addition, molecular dynamics and docking simulations confirmed, and further show, that the formation of an intramolecular disulfide bond, due to oxidative conditions, affects the CYGB 3D structure, thereby opening the access to the heme group, through gate functioning of His117. Altogether, the results obtained in this study (1) show that plasma-produced RONS can extensively oxidize proteins and (2) that the oxidation status of two redox-active cysteines lead to different conformations of CYGB.

Dietary Se supplementation partially restores the REDOX proteomic map of M. spretus liver exposed to p,p'-DDE.

The toxicity of p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), a contaminant and metabolite derivative of DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] is partially mediated by reactive oxygen species. Protein cysteine-based regulatory switches and subsequent alterations of the overall hepatic metabolism are triggered by p,p'-DDE through the disruption of the cellular redox status. The consequences are reproductive impairment, metabolic disorders, diabetes, neurotoxicity and cancer. In recent years, the risk of p,p'-DDE exposure has increased worldwide, reflecting the rise of mosquito-borne diseases in tropical countries that produce and export contaminated foods. Selenium (Se) is an essential trace element in animal nutrition with antioxidant properties that protects against the toxicity of some xenobiotics. We analyzed the ability of diet Se-supplementation to prevent damages induced by p,p'-DDE in the liver of M. spretus mice, by using redox proteomics based on the determination of the redox status of protein Cys residues. Se selectively acted on specific target, restoring the redox status and functionality of some membrane proteins involved in mitochondrial functionality, protein transport, cell signaling and protein metabolism. However, the Se-enriched diet did not completely prevent the metabolic shift caused by p,p'-DDE exposure that leads to disturbed lipogenesis, hepatic steatosis and alterations in the synthesis of hormones and other cell signals.