Unsaturated Aldehyde Acrolein Research Articles

ROCK1 Mediates Retinal Glial Cell Migration Promoted by Acrolein.

Objective: Acrolein is a highly reactive aldehyde that covalently binds to cellular macromolecules and subsequently modulates cellular function. Our previous study demonstrated that acrolein induces glial cell migration, a pathological hallmark of diabetic retinopathy; however, the detailed cellular mechanism remains unclear. The purpose of this study was to investigate the role of acrolein in retinal glial cell migration by focusing on rho-associated coiled-coil-containing protein kinases (ROCKs).Methods: Immunofluorescence staining for ROCK isoforms was performed using sections of fibrovascular tissue obtained from the eyes of patients with proliferative diabetic retinopathy (PDR). Rat retinal Müller glial cell line, TR-MUL5, was stimulated with acrolein and the levels of ROCK1 were evaluated using real-time PCR and western blotting. Phosphorylation of the myosin-binding subunit of myosin light chain phosphatase [myosin phosphatase target subunit 1, (MYPT1)] and myosin light chain 2 (MLC2) was assessed. The cell migration rate of TR-MUL5 cells exposed to acrolein and/or ripasudil, a non-selective ROCK inhibitor, was measured using the Oris cell migration assay.Results: ROCK isoforms, ROCK1 and ROCK2, were positively stained in the cytosol of glial cells in fibrovascular tissues. In TR-MUL5 cells, the mRNA expression level of Rock1, but not Rock2, was increased following acrolein stimulation. In line with the PCR data, western blotting showed increase in ROCK1 and cleaved ROCK1 protein in TR-MUL5 cells stimulated with acrolein. N-acetylcysteine (NAC) suppressed acrolein-associated Rock1 upregulation in TR-MUL5 cells. Acrolein augmented the phosphorylation of MYPT1 and MLC2 and increased the cell migration rate of TR-MUL5 cells, both of which were abrogated by ripasudil.Conclusions: Our study demonstrated that ROCK1 mediates the migration of retinal glial cells promoted by the unsaturated aldehyde acrolein.

Pathological Role of Unsaturated Aldehyde Acrolein in Diabetic Retinopathy.

With increasing prevalence of diabetes and a progressively aging society, diabetic retinopathy is emerging as one of the global leading causes of blindness. Recent studies have shown that vascular endothelial growth factor (VEGF) plays a central role in the pathogenesis of diabetic retinopathy and anti-VEGF agents have become the first-line therapy for the vision-threatening disease. However, recent studies have also demonstrated that diabetic retinopathy is a multifactorial disease and that VEGF-independent mechanism(s) also underlie much of the pathological changes in diabetic retinopathy. Acrolein is a highly reactive unsaturated aldehyde and is implicated in protein dysfunction. As acrolein is common in air pollutants, previous studies have focused on it as an exogenous causative factor, for instance, in the development of respiratory diseases. However, it has been discovered that acrolein is also endogenously produced and induces cell toxicity and oxidative stress in the body. In addition, accumulating evidence suggests that acrolein and/or acrolein-conjugated proteins are associated with the molecular mechanisms in diabetic retinopathy. This review summarizes the pathological roles and mechanisms of endogenous acrolein production in the pathogenesis of diabetic retinopathy.

Acrolein-Trapping Mechanism of Theophylline in Green Tea, Coffee, and Cocoa: Speedy and Successful.

Increasing evidence has identified the unsaturated aldehyde acrolein (ACR) as the potential factor that causes deoxyribonucleic acid cross-linking and the development of chronic diseases. The objective of this study was to investigate the mechanism by which theophylline (TP) scavenges ACR for the first time. TP efficiently scavenged ACR through forming adducts, which was demonstrated in a system in which TP was incubated with ACR at different ratios for different times for liquid chromatography with tandem mass spectrometry. Then, the mono- and di-ACR-TP adducts were purified, and their structures were elucidated by high-resolution mass spectrometry and nuclear magnetic resonance analysis. We found that the ACR residue on mono-ACR-TP further trapped one more ACR and formed di-ACR-TP adducts. Furthermore, mono- and di-ACR-TP had similar time-dependent ACR-scavenging activity to TP. Finally, we demonstrated that green tea, coffee, and cocoa inhibited ACR by trapping ACR to form mono- and di-ACR-TP adducts during the incubation of green tea, coffee, and cocoa with ACR.

Unsaturated Aldehyde Acrolein Promotes Retinal Glial Cell Migration.

To investigate the effect of the unsaturated aldehyde acrolein on retinal glial cell migration. Müller glial cell markers expression in TR-MUL5 were confirmed by RT-PCR and immunostaining. Cell viability and migration rate of TR-MUL5 cells were assessed after the stimulation with acrolein. DNA microarray analysis was performed to analyze changes in the expression levels of migration-related genes in Müller glial cells stimulated with acrolein. Real-time PCR and ELISA were performed to validate DNA microarray analysis results. Inhibitors of C-X-C motif chemokine ligand 1 (CXCL1), one of the genes highly upregulated after the exposure to acrolein, and blockers of its receptor, CXCR2, were used to investigate the role of the CXCL1-CXCR2 axis on glial cell migration. CXCL1 concentration was measured in vitreous fluid samples obtained from proliferative diabetic retinopathy (PDR) and nondiabetic control eyes. CXCL1 and CXCR2 expression in glial cells of fibrovascular tissues obtained from PDR patients was examined by immunostaining. At a high concentration, acrolein (100 μM) significantly decreased cell viability. However, in moderate, sublethal concentrations (25-50 μM), acrolein induced cell migration and substantially increased the production of CXCL1 in TR-MUL5 cells. CXCL1 concentration was significantly elevated in vitreous fluids of PDR patients, and CXCL1 and CXCR2 were present in glial cells in fibrovascular tissues of PDR patients. CXCL1 stimulation increased glial cell migration in a dose-dependent manner, which was abrogated by the neutralization of the CXCL1-CXCR2 axis. Our data demonstrate that acrolein promotes retinal Müller glial cell migration by enhancing CXCL1 production.

Soluble Vascular Adhesion Protein-1 Mediates Spermine Oxidation as Semicarbazide-Sensitive Amine Oxidase: Possible Role in Proliferative Diabetic Retinopathy

ABSTRACTPurpose/Aim of the study: To explore the possible role of vascular adhesion protein-1 (VAP-1) via its enzymatic function as a semicarbazide-sensitive amine oxidase (SSAO) in the pathogenesis of proliferative diabetic retinopathy (PDR).Materials and Methods: The levels of soluble VAP-1/SSAO and the unsaturated aldehyde acrolein (ACR)-conjugated protein, Nε-(3-formyl-3, 4-dehydropiperidino) lysine adduct (FDP-Lys), were measured in vitreous fluid samples of PDR and non-diabetic patients using ELISA. Recombinant human VAP-1/SSAO (rhVAP-1/SSAO) was incubated with spermine, with or without semicarbazide or RTU-1096 (a specific inhibitor for VAP-1/SSAO). Immunofluorescence assays were performed to assess the localization of VAP-1/SSAO and FDP-Lys in fibrovascular tissues from patients with PDR. The impact of ACR on cultured retinal capillary endothelial cells was assessed using a cell viability assay and total glutathione (GSH) measurements.Results: The levels of sVAP-1/SSAO and FDP-Lys were elevated in the vitreous fluid of patients with PDR. Incubation of rhVAP-1 with spermine resulted in the generation of hydrogen peroxide and FDP-Lys and the production was inhibited by semicarbazide and RTU-1096. In fibrovascular tissues, FDP-Lys and VAP-1/SSAO were present in endothelial cells. ACR stimulation reduced GSH levels in the cultured endothelial cells in a dose-dependent manner and caused cellular toxicity.Conclusions: Our results indicate the pathological role of sVAP-1/SSAO to generate hydrogen peroxide and toxic aldehyde ACR, both of which are associated with oxidative stress, as a consequence of spermine oxidation in eyes with PDR.

Acrolein increases macrophage atherogenicity in association with gut microbiota remodeling in atherosclerotic mice: protective role for the polyphenol-rich pomegranate juice.

The unsaturated aldehyde acrolein is pro-atherogenic, and the polyphenol-rich pomegranate juice (PJ), known for its anti-oxidative/anti-atherogenic properties, inhibits macrophage foam cell formation, the hallmark feature of early atherosclerosis. This study aimed to investigate two unexplored areas of acrolein atherogenicity: macrophage lipid metabolism and the gut microbiota composition. The protective effects of PJ against acrolein atherogenicity were also evaluated. Atherosclerotic apolipoprotein E-deficient (apoE-/-) mice that were fed acrolein (3mg/kg/day) for 1month showed significant increases in serum and aortic cholesterol, triglycerides, and lipid peroxides. In peritoneal macrophages isolated from the mice and in J774A.1 cultured macrophages, acrolein exposure increased intracellular oxidative stress and stimulated cholesterol and triglyceride accumulation via enhanced rates of their biosynthesis and over-expression of key regulators of cellular lipid biosynthesis: sterol regulatory element-binding proteins (SREBPs), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and diacylglycerol acyltransferase1 (DGAT1). Acrolein-fed mice demonstrated a major shift in the gut microbiota composition, including a significant phylum-level change in increased Firmicutes and decreased Bacteroidetes. At the family level, acrolein significantly increased the prevalence of Ruminococcaceae and Lachnospiraceae of which the Coprococcus genus was significantly and positively correlated with serum, aortic and macrophage lipid levels and peroxidation. The pro-atherogenic effects of acrolein on serum, aortas, macrophages, and the gut microbiota were substantially abolished by PJ. In conclusion, these findings provide novel mechanisms by which acrolein increases macrophage lipid accumulation and alters the gut microbiota composition in association with enhanced atherogenesis. Moreover, PJ was found as an effective strategy against acrolein atherogenicity.

Glutathione S-transferase P protects against cyclophosphamide-induced cardiotoxicity in mice

High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100–300mg/kg) increased plasma levels of creatine kinase-MB isoform (CK·MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null than WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein–acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1–5mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10–20mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity.

Acrolein, a highly toxic aldehyde generated under oxidative stress in vivo, aggravates the mouse liver damage after acetaminophen overdose.

Although acetaminophen-induced liver injury in mice has been extensively studied as a model of human acute drug-induced hepatitis, the mechanism of liver injury remains unclear. Liver injury is believed to be initiated by metabolic conversion of acetaminophen to the highly reactive intermediate N-acetyl p-benzoquinoneimine, and is aggravated by subsequent oxidative stress via reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and the hydroxyl radical (•OH). In this study, we found that a highly toxic unsaturated aldehyde acrolein, a byproduct of oxidative stress, has a major role in acetaminophen-induced liver injury. Acetaminophen administration in mice resulted in liver damage and increased acrolein-protein adduct formation. However, both of them were decreased by treatment with N-acetyl-L-cysteine (NAC) or sodium 2-mercaptoethanesulfonate (MESNA), two known acrolein scavengers. The specificity of NAC and MESNA was confirmed in cell culture, because acrolein toxicity, but not H2O2 or •OH toxicity, was inhibited by NAC and MESNA. These results suggest that acrolein may be more strongly correlated with acetaminophen-induced liver injury than ROS, and that acrolein produced by acetaminophen-induced oxidative stress can spread from dying cells at the primary injury site, causing damage to the adjacent cells and aggravating liver injury.

1-Cyano-2,3-epithiopropane is a novel plant-derived chemopreventive agent which induces cytoprotective genes that afford resistance against the genotoxic , -unsaturated aldehyde acrolein

Epithionitriles represent a previously unrecognized class of cancer chemopreventive phytochemical generated from alkenyl glucosinolates in cruciferous vegetables. In rat liver RL-34 epithelial cells, 1-cyano-2,3-epithiopropane (CETP), 1-cyano-3,4-epithiobutane (CETB) and 1-cyano-4,5-epithiopentane (CETPent) were shown to induce cytoprotective enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione (GSH) S-transferase A3 and the glutamate-cysteine ligase modifier subunit; CETP was more potent in this regard than were either CETB or CETPent, with 50 microM CETP eliciting a remarkable approximately 10-fold induction of NQO1. Furthermore, 50 microM CETP stimulated a 2.0-fold overproduction of GSH in RL-34 cells. Transfection experiments demonstrated that epithionitriles induced gene expression through an antioxidant response element (ARE) and that transactivation of an Nqo1-luciferase reporter plasmid was dependent on NF-E2 p45-related factor 2 (Nrf2), a cap'n'collar basic region leucine zipper transcription factor. Evidence is presented that CETP affected Nrf2-mediated induction of ARE-driven transcription by inhibiting Kelch-like ECH-associated protein 1 (Keap1), a ubiquitin ligase substrate adaptor that negatively regulates Nrf2. We found that Nqo1 was expressed constitutively at high levels in Keap1(-/-) mouse embryonic fibroblasts (MEFs) and it was not further induced by CETP. However, knock-in of mouse Keap1 or zebrafish Keap1a into Keap1(-/-) MEFs repressed Nqo1-luciferase reporter gene activity, but repression by the murine or zebrafish proteins was antagonized by CETP. Pre-treatment of Nrf2(+/+) MEFs, but not Nrf2(-/-) MEFs, with 15 microM CETP for 24 h conferred 2.4-fold resistance against subsequent exposure to the alpha,beta-unsaturated aldehyde acrolein, indicating that the phytochemical exerts chemopreventive properties against genotoxic xenobiotics.

Mechanisms of acrolein-induced myocardial dysfunction: implications for environmental and endogenous aldehyde exposure

Aldehydes are ubiquitous pollutants generated during the combustion of organic materials and are present in air, water, and food. Several aldehydes are also endogenous products of lipid peroxidation and by-products of drug metabolism. Despite well-documented high reactivity of unsaturated aldehydes, little is known regarding their cardiovascular effects and their role in cardiac pathology. Accordingly, we examined the myocardial effects of the model unsaturated aldehyde acrolein. In closed-chest mice, intravenous acrolein (0.5 mg/kg) induced rapid but reversible left ventricular dilatation and dysfunction. In mouse myocytes, micromolar acrolein acutely depressed myofilament Ca(2+) responsiveness without altering catecholamine sensitivity, similar to the phenotype of stunned myocardium. Immunoblotting revealed increased acrolein-protein adducts and protein-carbonyls in both acrolein-exposed myocardium (1.8-fold increase, P < 0.002) and myocytes (6.4-fold increase, P < 0.02). Both the contractile dysfunction and adduct formation were markedly attenuated by pretreatment with the thiol donor N-acetylcysteine (5 mM). Two-dimensional gel electrophoresis and mass-assisted laser desorption/ionization time-of-flight mass spectrometry analysis revealed two groups of adducted proteins, sarcomeric/cytoskeletal proteins (cardiac alpha-actin, desmin, myosin light polypeptide 3) and energy metabolism proteins (mitochondrial creatine kinase-2, ATP synthase), indicating site-specific protein modification that was confirmed by immunohistochemical colocalization. We conclude that direct exposure to acrolein induces selective myofilament impairment, which may be, in part, related to the modification of proteins involved in myocardial contraction and energy metabolism. Myocardial dysfunction induced by acrolein and related aldehydes may be symptomatic of toxicological states associated with ambient or occupational exposures or drug toxicity. Moreover, aldehydes such as acrolein may mediate cardiac dysfunction in pathologies characterized by high-oxidative stress.

Prostacyclin Prevents Pulmonary Endothelial Cell Apoptosis Induced by Cigarette Smoke

Impaired endothelial cell-dependent vasodilation, inflammation, apoptosis, and proliferation are manifestations of endothelial dysfunction in chronic obstructive pulmonary disease (COPD). Prostacyclin (PGI(2)) is a major product of the cyclooxygenase pathway with potent vasodilatory and antimitogenic properties and may be relevant to endothelial dysfunction in COPD. To determine if PGI(2) expression is altered in smoking-related lung disease and if it may be protective in COPD-associated endothelial dysfunction. We evaluated, by immunohistochemistry, Western blotting, and polymerase chain reaction, human emphysema tissue compared with normal tissue for expression of prostacyclin synthase (PGI(2)S). We examined the effects of cigarette smoke extract (CSE) and aldehyde components on eicosanoid expression in primary human pulmonary microvascular endothelial cells. Finally, we used a murine model of lung-specific PGI(2)S overexpression and in vitro studies to determine if PGI(2) expression has protective effects on cigarette smoke-induced endothelial apoptosis. Human emphysema lung tissue exhibited lower PGI(2)S expression within the pulmonary endothelium than in normal lung. In vitro studies demonstrated that CSE, and in particular the alpha,beta unsaturated aldehyde acrolein, suppressed PGI(2)S gene expression, whereas CSE significantly induced the upstream mediators COX-2 and cytosolic phospholipase A2 in human pulmonary microvascular endothelial cells. Mice with lung-specific PGI(2)S overexpression exhibited less endothelial apoptosis after chronic smoke exposure. In vitro, iloprost exhibited protective effects on CSE-induced apoptosis. PGI(2) has protective effects in the pulmonary vasculature after acute and chronic cigarette smoke exposure. An imbalance in eicosanoid expression may be important to COPD-associated endothelial dysfunction.

Effects of cigarette smoke components on in vitro chemotaxis of human polymorphonuclear leukocytes.

Some ciliostatic components of cigarette smoke were studied as inhibitors of in vitro chemotaxis of human polymorphonuclear leukocytes (PMNs). In comparison to their concentration in an inhibitory level of cigarette smoke, the unsaturated aldehydes acrolein and crotonaldehyde were the most potent inhibitors, whereas nicotine, cyanide, acetaldehyde, and furfural were the next strongest inhibitors. In contrast, sulfide, propionaldehyde, butyraldehyde, and the phenols (phenol and o-, m-, and p-cresol) were relatively weak inhibitors of PMN chemotaxis. Acrolein and crotonaldehyde mimicked whole cigarette smoke in their effects on PMNs by not causing loss of PMN viability, yet their effects were prevented by the addition of cysteine. On the other hand, addition of nicotine, cyanide, acetaldehyde, and furfural to PMN suspensions resulted in a limited loss of cellular viabilities, and their effects on PMNs were not prevented by cysteine. Of the tested components, only cyanide significantly altered PMN glucose metabolism by increasing carbon flow via the glycolytic and hexose monophosphate pathways in a manner similar to that observed with whole cigarette smoke. The results of this study suggest that the unsaturated aldehydes, including acrolein and crotonaldehyde, are major contributors to the inhibitory properties of cigarette smoke. The inhibitory effects of these unsaturated aldehydes are probably due to a direct interaction of these oxidants and/or thiol-alkylating agents with PMNs, yet the glucose metabolism of these cells is unaffected. One interpretation of these data is that PMN chemotaxis is dependent upon particular cellular proteins containing one or more essential thiol group(s) but that these proteins are unrelated to glucose metabolism.

Acrolein Polymers

AbstractThe unsaturated aldehyde acrolein is a suitable source for different types of high polymers. If it is regarded as a 1,3‐diene analogous to butadiene, then all the known modes of its polymerization can be systematically described and further possibilities predicted. A survey is given on this basis of the results obtained during the past decade. It is shown that acrolein is one of the few monomers which contain two polymerizable groups of differing reactivities. This fact allows the formation from acrolein of new homopolymers, copolymers, and graft copolymers, a scope which is not encountered with many other monomers.