Extracellularly secreted cysteine derived from cystine regulates oxidative and electrophilic stress in HepG2 cells.

While cysteine (CysSH) is known to be exported into the extracellular space, its biological significance is not well understood. The present study examined the movement of extracellular CysSH using stable isotope-labeled cystine (CysSSCys), which is transported into cells and reduced to CysSH. Exposure of HepG2 cells to 100 µM stable isotope-labeled CysSSCys resulted in 70 µM labeled CysSH in cell medium 1 h after CysSSCys exposure. When the cell medium was collected and incubated with either hydrogen peroxide (H2O2) or atmospheric electrophiles, such as 1,2-naphthoquinone, 1,4-naphthoquinone and 1,4-benzoquinone, CysSH in the cell medium was almost completely consumed. In contrast, extracellular levels of CysSH were unaltered during exposure of HepG2 cells to H2O2 for up to 2 h, suggesting redox cycling of CysSSCys/CysSH in the cell system. Experiments with and without changing cell medium containing CysSH from HepG2 cells revealed that oxidative and electrophilic modifications of cellular proteins, caused by exposure to H2O2 and 1,2-naphthoquinone, were significantly repressed by CysSH in the medium. We also examined participation of enzymes and/or antioxidants in intracellular reduction of CysSSCys to CysSH. These results provide new findings that extracellular CysSH derived from CysSSCys plays a role in the regulation of oxidative and electrophilic stress.

Lipid-derived electrophiles inhibit the function of membrane channels during ferroptosis.

The therapeutic targeting of ferroptosis requires full understanding of the molecular mechanism of this regulated cell death pathway. While lipid-derived electrophiles (LDEs), including 4-hydroxy-2-nonenal (4-HNE), are important biomarkers of ferroptosis, a functional role for these highly reactive species in ferroptotic cell death execution has not been established. Here, through mechanistic characterization of LDE-detoxification impairment, we demonstrate that LDEs mediate altered protein function during ferroptosis. Applying live cell fluorescence imaging, we first identified that export of glutathione-LDE-adducts through multidrug resistance-associated protein (MRP) channels is inhibited following exposure to a panel of ferroptosis inducers (FINs) with different modes of action (type I-IV FINs erastin, RSL3, FIN56, and FINO2). This channel inhibition was recreated by both initiation of lipid peroxidation and treatment with 4-HNE. Importantly, treatment with radical-trapping antioxidants prevented impaired LDE-adduct export when working with both FINs and lipid peroxidation initiators but not 4-HNE, pinpointing LDEs as the cause of this inhibited MRP activity observed during ferroptosis. Our findings, when combined with reports of widespread LDE alkylation of key proteins following ferroptosis induction, including MRP1, set a precedent for LDEs as critical mediators of ferroptotic cell damage. Lipid hydroperoxide breakdown to form truncated phospholipids and LDEs may fully explain membrane permeabilization and modified protein function downstream of lipid peroxidation, offering a unified explanation of the molecular cell death mechanism of ferroptosis.

Vimentin Tail Segments Are Differentially Exposed at Distinct Cellular Locations and in Response to Stress.

The intermediate filament protein vimentin plays a key role in cell signaling and stress sensing, as well as an integrator of cytoskeletal dynamics. The vimentin monomer consists of a central rod-like domain and intrinsically disordered head and tail domains. Although the organization of vimentin oligomers in filaments is beginning to be understood, the precise disposition of the tail region remains to be elucidated. Here we observed that electrophilic stress-induced condensation shielded vimentin from recognition by antibodies against specific segments of the tail domain. A detailed characterization revealed that vimentin tail segments are differentially exposed at distinct subcellular locations, both in basal and stress conditions. The 411-423 segment appeared accessible in all cell areas, correlating with vimentin abundance. In contrast, the 419-438 segment was more scantily recognized in perinuclear vimentin and lipoxidative stress-induced bundles, and better detected in peripheral filaments, where it appeared to protrude further from the filament core. These differences persisted in mitotic cells. Interestingly, both tail segments showed closer accessibility in calyculin A-treated cells and phosphomimetic mutants of the C-terminal region. Our results lead us to hypothesize the presence of at least two distinct arrangements of vimentin tail in cells: an "extended" conformation (accessible 419-438 segment), preferentially detected in peripheral areas with looser filaments, and a "packed" conformation (shielded 419-438 segment), preferentially detected at the cell center in robust filaments and lipoxidative stress-induced bundles. These different arrangements could be putatively interconverted by posttranslational modifications, contributing to the versatility of vimentin functions and/or interactions.

The Old Yellow Enzyme OfrA Fosters Staphylococcus aureus Survival via Affecting Thiol-Dependent Redox Homeostasis.

Old yellow enzymes (OYEs) are widely found in the bacterial, fungal, and plant kingdoms but absent in humans and have been used as biocatalysts for decades. However, OYEs' physiological function in bacterial stress response and infection situations remained enigmatic. As a pathogen, the Gram-positive bacterium Staphylococcus aureus adapts to numerous stress conditions during pathogenesis. Here, we show that in S. aureus genome, two paralogous genes (ofrA and ofrB) encode for two OYEs. We conducted a bioinformatic analysis and found that ofrA is conserved among all publicly available representative staphylococcal genomes and some Firmicutes. Expression of ofrA is induced by electrophilic, oxidative, and hypochlorite stress in S. aureus. Furthermore, ofrA contributes to S. aureus survival against reactive electrophilic, oxygen, and chlorine species (RES, ROS, and RCS) via thiol-dependent redox homeostasis. At the host-pathogen interface, S. aureusΔofrA has defective survival in macrophages and whole human blood and decreased staphyloxanthin production. Overall, our results shed the light onto a novel stress response strategy in the important human pathogen S. aureus.

Chemically Tuned, Reversible Fluorogenic Electrophile for Live Cell Nanoscopy.

We report a chemically tuned fluorogenic electrophile designed to conduct live-cell super-resolution imaging by exploiting its stochastic reversible alkylation reaction with cellular nucleophiles. Consisting of a lipophilic BODIPY fluorophore tethered to an electrophilic cyanoacrylate warhead, the new probe cyanoAcroB remains nonemissive due to internal conversion along the cyanoacrylate moiety. Intermittent fluorescence occurs following thiolate Michael addition to the probe, followed by retro-Michael reaction, tuned by the cyano moiety in the acrylate warhead and BODIPY decoration. This design enables long-term super-resolved imaging of live cells by preventing fluorescent product accumulation and background increase, while preserving the pool of the probe. We demonstrate the imaging capabilities of cyanoAcroB via two methods: (i) single-molecule localization microscopy imaging with nanometer accuracy by stochastic chemical activation and (ii) super-resolution radial fluctuation. The latter tolerates higher probe concentrations and low imaging powers, as it exploits the stochastic adduct dissociation. Super-resolved imaging with cyanoAcroB reveals that electrophile alkylation is prevalent in mitochondria and endoplasmic reticulum. The 2D dynamics of these organelles within a single cell are unraveled with tens of nanometers spatial and sub-second temporal resolution through continuous imaging of cyanoAcroB extending for tens of minutes. Our work underscores the opportunities that reversible fluorogenic probes with bioinspired warheads bring toward illuminating chemical reactions with super-resolved features in live cells.

Cysteine trisulfide oxidizes protein thiols and induces electrophilic stress in human cells.

The cellular effects of hydrogen sulfide (H2S) signaling may be partially mediated by the formation of alkyl persulfides from thiols, such as glutathione and protein cysteine residues. Persulfides are potent nucleophiles and reductants and therefore potentially an important endogenous antioxidant or protein post-translational modification. To directly study the cellular effects of persulfides, cysteine trisulfide (Cys-S3) has been proposed as an in situ persulfide donor, as it reacts with cellular thiols to generate cysteine persulfide (Cys-S-S-). Numerous pathways sense and respond to electrophilic cellular stressors to inhibit cellular proliferation and induce apoptosis, however the effect of Cys-S3 on the cellular stress response has not been addressed. Here we show that Cys-S3 inhibited cellular metabolism and proliferation and rapidly induced cellular- and ER-stress mechanisms, which were coupled to widespread protein-thiol oxidation. Cys-S3 reacted with Na2S to generate cysteine persulfide, which protected human cell lines from ER-stress. However this method of producing cysteine persulfide contains excess sulfide, which interferes with the direct analysis of persulfide donation. We conclude that cysteine trisulfide is a thiol oxidant that induces cellular stress and decreased proliferation.

A comparison of the chemical biology of hydropersulfides (RSSH) with other protective biological antioxidants and nucleophiles.

The hydropersulfide (RSSH) functional group has received significant recent interest due to its unique chemical properties that set it apart from other biological species. The chemistry of RSSH predicts that one possible biological role may be as a protectant against cellular oxidative and electrophilic stress. That is, RSSH has reducing and nucleophilic properties that may combat the potentially destructive biochemistry of toxicologically relevant oxidants and electrophiles. However, there are currently numerous other molecules that have established roles in this regard. For example, ascorbate and tocopherols are potent antioxidants that quench deleterious oxidative reactions and glutathione (GSH) is a well-established and highly prevalent biological protectant against electrophile toxicity. Thus, in order to begin to understand the possible role of RSSH species as protectants against oxidative/electrophilic stress, the inherent chemical properties of RSSH versus these other protectants will be discussed and contrasted.

Landscape of electrophilic and inflammatory stress-mediated gene regulation in human lymphoblastoid cell lines.

Human lymphoblastoid cell lines (LCLs) are valuable for the functional analyses of diseases. We have established more than 4200 LCLs as one of the resources of an integrated biobank. While oxidative and inflammatory stresses play critical roles in the onset and progression of various diseases, the responsiveness of LCLs, especially that of biobank-made LCLs, to these stresses has not been established. To address how LCLs respond to these stresses, in this study, we performed RNA sequencing of eleven human LCLs that were treated with an electrophile, diethyl maleate (DEM) and/or an inflammatory mediator, lipopolysaccharide (LPS). We found that over two thousand genes, including those regulated by a master regulator of the electrophilic/oxidative stress response, NRF2, were upregulated in LCLs treated with DEM, while approximately three hundred genes, including inflammation-related genes, were upregulated in LPS-treated LCLs. Of the LPS-induced genes, a subset of proinflammatory genes was repressed by DEM, supporting the notion that DEM suppresses the expression of proinflammatory genes through NRF2 activation. Conversely, a part of DEM-induced gene was repressed by LPS, suggesting reciprocal interference between electrophilic and inflammatory stress-mediated pathways. These data clearly demonstrate that LCLs maintain, by and large, responsive pathways against oxidative and inflammatory stresses and further endorse the usefulness of the LCL supply from the biobank.

Reactive Chemicals and Electrophilic Stress in Cancer: A Minireview.

Exogenous reactive chemicals can impair cellular homeostasis and are often associated with the development of cancer. Significant progress has been achieved by studying the macromolecular interactions of chemicals that possess various electron-withdrawing groups and the elucidation of the protective responses of cells to chemical interventions. However, the formation of electrophilic species inside the cell and the relationship between oxydative and electrophilic stress remain largely unclear. Derivatives of nitro-benzoxadiazole (also referred as nitro-benzofurazan) are potent producers of hydrogen peroxide and have been used as a model to study the generation of reactive species in cancer cells. This survey highlights the pivotal role of Cu/Zn superoxide dismutase 1 (SOD1) in the production of reactive oxygen and electrophilic species in cells exposed to cell-permeable chemicals. Lipophilic electrophiles rapidly bind to SOD1 and induce stable and functionally active dimers, which produce excess hydrogen peroxide leading to aberrant cell signalling. Moreover, reactive oxygen species and reactive electrophilic species, simultaneously generated by redox reactions, behave as independent entities that attack a variety of proteins. It is postulated that the binding of the electrophilic moiety to multiple proteins leading to impairing different cellular functions may explain unpredictable side effects in patients undergoing chemotherapy with reactive oxygen species (ROS)-inducing drugs. The identification of proteins susceptible to electrophiles at early steps of oxidative and electrophilic stress is a promising way to offer rational strategies for dealing with stress-related malignant tumors.

Oxidized phospholipids stimulate production of stem cell factor via NRF2-dependent mechanisms.

Receptor tyrosine kinase c-Kit and its ligand stem cell factor (SCF) regulate resident vascular wall cells and recruit circulating progenitors. We tested whether SCF may be induced by oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) known to accumulate in atherosclerotic vessels. Gene expression analysis demonstrated OxPAPC-induced upregulation of SCF mRNA and protein in different types of endothelial cells (ECs). Elevated levels of SCF mRNA were observed in aortas of ApoE-/- knockout mice. ECs produced biologically active SCF because conditioned medium from OxPAPC-treated cells stimulated activation (phosphorylation) of c-Kit in naïve ECs. Induction of SCF by OxPAPC was inhibited by knocking down transcription factor NRF2. Inhibition or stimulation of NRF2 by pharmacological or molecular tools induced corresponding changes in SCF expression. Finally, we observed decreased levels of SCF mRNA in aortas of NRF2 knockout mice. We characterize OxPLs as a novel pathology-associated stimulus inducing expression of SCF in endothelial cells. Furthermore, our data point to transcription factor NRF2 as a major mediator of OxPL-induced upregulation of SCF. This mechanism may represent one of the facets of pleiotropic action of NRF2 in vascular wall.

Toxic Electrophiles Induce Expression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas aeruginosa through a Novel Transcriptional Regulator, CmrA.

The multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa However, in so-called nfxC mutants, mutational alteration of the gene mexS results in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA (for chloramphenicol resistance activator). In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprN through MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosa to the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.

The chemical biology of protein hydropersulfides: Studies of a possible protective function of biological hydropersulfide generation.

The recent discovery of significant hydropersulfide (RSSH) levels in mammalian tissues, fluids and cells has led to numerous questions regarding their possible physiological function. Cysteine hydropersulfides have been found in free cysteine, small molecule peptides as well as in proteins. Based on their chemical properties and likely cellular conditions associated with their biosynthesis, it has been proposed that they can serve a protective function. That is, hydropersulfide formation on critical thiols may protect them from irreversible oxidative or electrophilic inactivation. As a prelude to understanding the possible roles and functions of hydropersulfides in biological systems, this study utilizes primarily chemical experiments to delineate the possible mechanistic chemistry associated with cellular protection. Thus, the ability of hydropersulfides to protect against irreversible electrophilic and oxidative modification was examined. The results herein indicate that hydropersulfides are very reactive towards oxidants and electrophiles and are modified readily. However, reduction of these oxidized/modified species is facile generating the corresponding thiol, consistent with the idea that hydropersulfides can serve a protective function for thiol proteins.

Look into brain energy crisis and membrane pathophysiology in ischemia and reperfusion.

In an ischemic environment, brain tissue responds to oxygen deprivation with the initiation of rapid changes in bioenergetic metabolism to ensure ion and metabolic homeostasis. At the same time, the accelerated cleavage of membrane phospholipids changes membrane composition and increases free fatty acid concentration. Phospholipid breakdown also generates specific messengers that participate in signaling cascades that can either promote neuronal protection or cause injury. The net impact of signaling events affects the final outcome of the stroke. While reoxygenation is a life-saving intervention, it can exacerbate brain damage. Although compromised energy metabolism is restored shortly after reperfusion, alterations in membrane phospholipid composition with subsequent accumulation of lipid oxoderivates are neurotoxic, causing oxidative stress and ischemia-reperfusion (IR) injury. Thus, plasma and mitochondrial membranes are the first responders as well as mediators of IR-induced stress signals. In this review, we focus on ischemia-induced changes in brain energy metabolism and membrane functions as the causal agents of cell stress responses upon reoxygenation. The first part of the review deals with the specificities of neuronal bioenergetics during IR and their impact on metabolic processes. The second part is concentrated on involvement of both plasma and mitochondrial membranes in the production of messengers which can modulate neuroprotective pathways or participate in oxidative/electrophilic stress responses. Although the etiology of IR injury is multifactorial, deciphering the role of membrane and membrane-associated processes in brain damage will uncover new therapeutic agents with the ability to stabilize neuronal membranes and modulate their responses in favor of prosurvival pathways.

On the role of 4-hydroxynonenal in health and disease.

Polyunsaturated fatty acids are susceptible to peroxidation and they yield various degradation products, including the main α,ß-unsaturated hydroxyalkenal, 4-hydroxy-2,3-trans-nonenal (HNE) in oxidative stress. Due to its high reactivity, HNE interacts with various macromolecules of the cell, and this general toxicity clearly contributes to a wide variety of pathological conditions. In addition, growing evidence suggests a more specific function of HNE in electrophilic signaling as a second messenger of oxidative/electrophilic stress. It can induce antioxidant defense mechanisms to restrain its own production and to enhance the cellular protection against oxidative stress. Moreover, HNE-mediated signaling can largely influence the fate of the cell through modulating major cellular processes, such as autophagy, proliferation and apoptosis. This review focuses on the molecular mechanisms underlying the signaling and regulatory functions of HNE. The role of HNE in the pathophysiology of cancer, cardiovascular and neurodegenerative diseases is also discussed.

Steatosis-induced proteins adducts with lipid peroxidation products and nuclear electrophilic stress in hepatocytes.

Accumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA) complex (1mM, 2:1 oleic and palmitic acids). In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS) was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2). Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation) with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP). 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment.

Protracted Oxidative Alterations in the Mechanism of Hematopoietic Acute Radiation Syndrome.

The biological effects of high-dose total body ionizing irradiation [(thereafter, irradiation (IR)] are attributed to primary oxidative breakage of biomolecule targets, mitotic, apoptotic and necrotic cell death in the dose-limiting tissues, clastogenic and epigenetic effects, and cascades of functional and reactive responses leading to radiation sickness defined as the acute radiation syndrome (ARS). The range of remaining and protracted injuries at any given radiation dose as well as the dynamics of post-IR alterations is tissue-specific. Therefore, functional integrity of the homeostatic tissue barriers may decline gradually within weeks in the post-IR period culminating with sepsis and failure of organs and systems. Multiple organ failure (MOF) leading to moribundity is a common sequela of the hemotapoietic form of ARS (hARS). Onset of MOF in hARS can be presented as "two-hit phenomenon" where the "first hit" is the underlying consequences of the IR-induced radiolysis in cells and biofluids, non-septic inflammation, metabolic up-regulation of pro-oxidative metabolic reactions, suppression of the radiosensitive hematopoietic and lymphoid tissues and the damage to gut mucosa and vascular endothelium. While the "second hit" derives from bacterial translocation and spread of the bacterial pathogens and inflammagens through the vascular system leading to septic inflammatory, metabolic responses and a cascade of redox pro-oxidative and adaptive reactions. This sequence of events can create a ground for development of prolonged metabolic, inflammatory, oxidative, nitrative, and carbonyl, electrophilic stress in crucial tissues and thus exacerbate the hARS outcomes. With this perspective, the redox mechanisms, which can mediate the IR-induced protracted oxidative post-translational modification of proteins, oxidation of lipids and carbohydrates and their countermeasures in hARS are subjects of the current review. Potential role of ubiquitous, radioresistant mesenchymal stromal cells in the protracted responses to IR and IR-related septicemia is also discussed.

Tissue sensitivity of the rat upper and lower extrapulmonary airways to the inhaled electrophilic air pollutants diacetyl and acrolein.

The target site for inhaled vapor-induced injury often differs in mouth-breathing humans compared with nose-breathing rats, thus complicating the use of rat inhalation toxicity data for assessment of human risk. We sought to examine sensitivity of respiratory/transitional nasal (RTM) and tracheobronchial (TBM) mucosa to two electrophilic irritant vapors: diacetyl and acrolein. Computational fluid dynamic physiologically based pharmacokinetic modeling was coupled with biomarker assessment to establish delivered dose-response relationships in RTM and TBM in male F344 rats following 6 h exposure to diacetyl or acrolein. Biomarkers included glutathione status, proinflammatory and antioxidant gene mRNA levels, and nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Modeling revealed that 0.0094-0.1653 µg acrolein/min-cm(2) and 3.9-21.6 µg diacetyl/min-cm(2) were deposited into RTM/TBM. Results indicate RTM and TBM were generally of similar sensitivity to diacetyl and acrolein. For instance, both tissues displayed induction of antioxidant and proinflammatory genes, and nuclear accumulation of Nrf2 after electrophile exposure. Hierarchical cellular response patterns were similar in RTM and TBM but differed between vapors. Specifically, diacetyl exposure induced proinflammatory and antioxidant genes concomitantly at low exposure levels, whereas acrolein induced antioxidant genes at much lower exposure levels than that required to induce proinflammatory genes. Generally, diacetyl was less potent than acrolein, as measured by maximal induction of transcripts. In conclusion, the upper and lower extrapulmonary airways are of similar sensitivity to inhaled electrophilic vapors. Dosimetrically based extrapolation of nasal responses in nose-breathing rodents may provide an approach to predict risk to the lower airways of humans during mouth-breathing.

MicroRNA miR-320a modulates induction of HO-1, GCLM and OKL38 by oxidized phospholipids in endothelial cells.

OBJECTIVE: Oxidized phospholipids (OxPLs), which are highly abundant in atherosclerotic lesions, are known to induce electrophilic stress response (ESR). ESR induces cytoprotective genes via the NF-E2-related factor 2 (NRF2) transcription factor. In order to get further insight into the mechanisms of ESR, we studied the role of microRNA (miR)-320a in induction of NRF2-dependent genes by OxPLs. METHODS: Microarray profiling and qRT-PCR methods were used for measurements of mRNA and miRNA levels. miR-320a levels were changed by transfection with synthetic oligonucleotides. Protein analysis was performed by Western blotting. The functional activity of NRF2 was measured by DNA-binding ELISA. RESULTS: Oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) induced miR-320a in endothelial cells. Induction of HO-1, OKL38 and GCLM mRNAs by OxPAPC and sulforaphane was attenuated upon knockdown of miR-320a. In contrast, transfection of ECs with miR-320a mimic oligonucleotide potentiated the effects of OxPAPC and sulforaphane on induction of HO-1, OKL38 and GCLM mRNAs. OxPAPC-induced p38 activation, levels of NRF2 protein and its ability to bind to consensus NRF2 DNA binding site were elevated in ECs transfected with miR-320a mimic. miR-320a positively regulated induction of VEGF mRNA by OxPAPC. Levels of miR-320a and HO-1 and OKL38 mRNAs were elevated in aortas of ApoE knockout mice fed with high fat diet. Manipulation of miR-320a level in ECs did not affect ability of OxPAPC to induce IL-8, COX-2 and MCP-1. CONCLUSION: miR-320a plays important role in induction of expression of HO-1, GCLM and OKL38 upon ESR induced either by OxPAPC or sulforaphane. These observations propose a general role of miR-320a in control of ESR induced by different electrophilic agents.