Melatonin prevents hypochlorous acid‐mediated cyanocobalamin destruction and cyanogen chloride generation

Hypochlorous acid (HOC l) is a potent cytotoxic oxidant generated by the enzyme myeloperoxidase (MPO ) in the presence of hydrogen peroxide (H₂O₂) and chloride (Cl^?). Elevated levels of HOC l play an important role in various pathological conditions through oxidative modification of several biomolecules. Recently, we have highlighted the ability of HOC l to mediate the destruction of the metal‐ion derivatives of tetrapyrrole macrocyclic rings such as hemoproteins and vitamin B₁₂ (VB ₁₂) derivatives. Destruction of cyanocobalamin, a common pharmacological form of VB ₁₂ mediated by HOC l, results in the generation of toxic molecular products such as chlorinated derivatives, corrin ring cleavage products, the toxic blood agents cyanide (CN ^?) and cyanogen chloride (CNC l), and redox‐active free cobalt. Here, we show that melatonin prevents HOC l‐mediated cyanocobalamin destruction, using a combination of UV ‐Vis spectrophotometry, high‐performance liquid chromatography analysis, and colorimetric CNC l assay. Identification of several melatonin oxidation products suggests that the protective role of melatonin against HOC l‐mediated cyanocobalamin destruction and subsequent CNC l generation is at the expense of melatonin oxidation. Collectively, this work highlights that, in addition to acting as an antioxidant and as a MPO inhibitor, melatonin can also prevent VB ₁₂ deficiency in inflammatory conditions such as cardiovascular and neurodegenerative diseases, among many others.     

Reaction of melatonin with hemoglobin-derived oxoferryl radicals and inhibition of the hydroperoxide-induced hemoglobin denaturation in red blood cells

Melatonin has been shown to act as a radical scavenger in various chemical and biological model systems in vitro. Kinetic evidence is now provided showing that melatonin inhibits the irreversible degradation of hemoglobin (Hb), when incubated with red blood cells exposed to the oxidant activity of cumene hydroperoxide (cumOOH). A decrease of heme loss and accumulation of soluble methemoglobin (met-Hb) are explained in terms of the interaction of the indoleamine with perferryl Hb (Hb[Fe(IV)=O]), a highly reactive Hb-derived radical species responsible for the irreversible Hb degradation. A kinetic study, in pure chemical solution, showed that melatonin can effectively reduce the oxoferryl heme group of perferryl-Hb, thus forming met-Hb. The reducing activity of melatonin is of the same order as that of Trolox, the water-soluble vitamin E analog. This novel radical-scavenging activity of melatonin may contribute to the previously observed protective effects of melatonin in ischemia-reperfusion injury.     

Oxidative reactions of normal and abnormal hemoglobins in the presence of phosphatidylserine vesicles

We have monitored a sequence of reactions involving hemoglobin (Hb) oxidation in the presence of phospholipid bilayers in carefully characterized buffer systems in order to delineate molecular events that may have cytotoxic effects, some of which may even lead to cellular death of erythrocytes. We followed reactions of subunit cross-linking, heme iron oxidation, heme destruction and iron release in normal (Hb A) and sickle [Hb S or beta6(A3)Glu-->Val] Hbs under different experimental conditions. Our results show that, in the presence of lipid surfaces, the reaction rates for heme Fe+2 to Fe+3 oxidation in Hb A and Hb S molecules were both enhanced by lipid surfaces. However, the extent of the enhancement in Hb A and Hb S was quite different in T5K6.5, a Tris buffer with low ionic strength and low pH. In T5K6.5, the rate constants were 0.8 h(-1) for Hb A and 4.3 h(-1) for Hb S, a 5-fold difference. This finding supports a published suggestion that sickle Hb exhibits abnormal heme oxidation in erythrocytes of sickle cell disease patients. We found that the rates were quite similar in P110N7.4, a phosphate buffer near physiological conditions. Even in T5K6.5, the lipid surface did not enhance Hb S reactions involving subunit cross-linking, heme destruction, or iron release. These findings suggest that heme oxidation and related reactions may not be responsible for detrimental cellular events in sickle erythrocytes under physiological conditions, as suggested in the literature.     

Oxidation of melatonin by taurine chloramine

Abstract: Melatonin is widely known for its antioxidant, immunomodulatory, and anti‐inflammatory effects. Hypochlorous acid (HOCl) is one example of an endogenous oxidant that is promptly neutralized by melatonin. Melatonin also inhibits myeloperoxidase, the enzyme that catalyzes the oxidation of chloride to HOCl. Taurine is the most abundant free amino acid in leukocytes. In activated neutrophils, taurine is converted to taurine chloramine (Tau‐NHCl) through a reaction with HOCl. In addition, the related compound taurine bromamine (Tau‐NHBr) can be released by neutrophils and eosinophils. The aim of this study was to investigate the reactivity of Tau‐NHCl and Tau‐NHBr with melatonin. We found that melatonin can react with either Tau‐NHCl or Tau‐NHBr, leading to the production of 2‐hydroxymelatonin and N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK). The reaction was pH‐dependent, and it occurs more rapidly at a slightly acidic pH. Tau‐NHBr was significantly more reactive than Tau‐NHCl. Using Tau‐NHBr as the oxidizing agent, 1 mm melatonin was oxidized in less than 1 min. The pH dependence of the reaction with Tau‐NHCl and the increased reactivity of Tau‐NHBr can be explained by a mechanism based on the initial attack of chloronium (Cl⁺) or bromonium (Br⁺) ions on melatonin. We also found that the addition of iodide to the reaction medium increased the yield of AFMK. These findings could contribute to the establishment of new functions for melatonin in inflammatory and parasitic diseases, where the role of this indoleamine has been extensively investigated.     

Neutrophil myeloperoxidase revisited: it's role in health and disease

Human myeloperoxidase (MPO; EC 1.11.1.7) is a specific heme (Fe³⁺) peroxidase, present in high concentrations in the azurophilic granules of neutrophils. Its amino acid and genomic sequences have been elucidated, and recombinant MPO is produced from genetically engineered mammalian cells. This peroxidase has the unique activity of chlorination, generating hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion, but also chlorine and monoatomic chlorine. By interacting with other enzymes of neutrophils and reacting with the products of neutrophil activation, MPO also produces other reactive oxygen species (singlet oxygen, hydroxyl radical, nitrosyl and nitryl chloride, etc.). In phagolysosomes, MPO acts together with NADPH oxidase and proteases for the destruction of the ingested organisms, by binding to the microorganism walls and producing locally HOCl, which is particularly active against the polysaccharidic capsules. MPO activity influences the transduction of the cellular signal (activation of NF-κB, chlorination of tyrosyl residues on essential enzymes, etc.) and modulates the functions of cells: it decreases the killer activity of NT lymphocytes and, after internalization, it enhances the microbial activity of macrophages. MPO is taken up by endothelial cells. MPO deficiency is the most common neutrophilic lysosomal enzyme deficiency, but usually without apparent increased susceptibility to infection or altered inflammatory response. MPO has been recognized to be responsible for the oxidation and chlorination of low density lipoproteins, contributing to the early stage of atherosclerosis. In disease with excessive and uncontrolled inflammatory reaction, MPO can be released in the extracellular milieu where it becomes cytotoxic for neighboring cells (oxidant stress) and oxidizes tissues and proteins (thiol oxidation, oxidation and chlorination of lipids and amino acids, etc.). Out of the neutrophil, the activity of MPO would be quickly inhibited by proteins; however, active MPO has been measured in broncho-alveolar lavage fluids from patients with acute lung injury. This specific enzyme, thus, presents a double role of essential host protection when acting into the phagocytes and of host damage when released in the extracellular milieu.     

Neutrophil myeloperoxidase revisited: it's role in health and disease

Summary Human myeloperoxidase (MPO; EC 1.11.1.7) is a specific heme (Fe³⁺) peroxidase, present in high concentrations in the azurophilic granules of neutrophils. Its amino acid and genomic sequences have been elucidated, and recombinant MPO is produced from genetically engineered mammalian cells. This peroxidase has the unique activity of chlorination, generating hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion, but also chlorine and monoatomic chlorine. By interacting with other enzymes of neutrophils and reacting with the products of neutrophil activation, MPO also produces other reactive oxygen species (singlet oxygen, hydroxyl radical, nitrosyl and nitryl chloride, etc.). In phagolysosomes, MPO acts together with NADPH oxidase and proteases for the destruction of the ingested organisms, by binding to the microorganism walls and producing locally HOCl, which is particularly active against the polysaccharidic capsules. MPO activity influences the transduction of the cellular signal (activation of NF-κB, chlorination of tyrosyl residues on essential enzymes, etc.) and modulates the functions of cells: it decreases the killer activity of NT lymphocytes and, after internalization, it enhances the microbial activity of macrophages. MPO is taken up by endothelial cells. MPO deficiency is the most common neutrophilic lysosomal enzyme deficiency, but usually without apparent increased susceptibility to infection or altered inflammatory response. MPO has been recognized to be responsible for the oxidation and chlorination of low density lipoproteins, contributing to the early stage of atherosclerosis. In disease with excessive and uncontrolled inflammatory reaction, MPO can be released in the extracellular milieu where it becomes cytotoxic for neighboring cells (oxidant stress) and oxidizes tissues and proteins (thiol oxidation, oxidation and chlorination of lipids and amino acids, etc.). Out of the neutrophil, the activity of MPO would be quickly inhibited by proteins; however, active MPO has been measured in broncho-alveolar lavage fluids from patients with acute lung injury. This specific enzyme, thus, presents a double role of essential host protection when acting into the phagocytes and of host damage when released in the extracellular milieu. Eingegangen: 3. September 1998 Akzeptiert: 4. Januar 1999     

Melatonin prevents hemorrhagic shock‐induced liver injury in rats through an Akt‐dependent HO‐1 pathway

Abstract: Although melatonin treatment following trauma‐hemorrhage or ischemic reperfusion prevents organs from dysfunction and injury, the precise mechanism remains unknown. This study tested whether melatonin prevents liver injury following trauma‐hemorrhage involved the protein kinase B (Akt)‐dependent heme oxygenase (HO)‐1 pathway. After a 5‐cm midline laparotomy, male rats underwent hemorrhagic shock (mean blood pressure approximately 40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, melatonin (2 mg/kg), or melatonin plus phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin (1 mg/kg). At 2 hr after trauma‐hemorrhage, the liver tissue myeloperoxidase activity, malondialdehyde, adenosine triphosphate, serum alanine aminotransferase, and aspartate aminotransferase levels were significantly increased compared with sham‐operated control. Trauma‐hemorrhage resulted in a significant decrease in the Akt activation in comparison with the shams (relative density, 0.526 ± 0.031 versus 1.012 ± 0.066). Administration of melatonin following trauma‐hemorrhage normalized liver Akt phosphorylation (0.993 ± 0.061), further increased mammalian target of rapamycin (mTOR) activation (5.263 ± 0.338 versus 2.556 ± 0.225) and HO‐1 expression (5.285 ± 0.325 versus 2.546 ± 0.262), and reduced cleaved caspase‐3 levels (2.155 ± 0.297 versus 5.166 ± 0.309). Coadministration of wortmannin abolished the melatonin‐mediated attenuation of the shock‐induced liver injury markers. Our results collectively suggest that melatonin prevents hemorrhagic shock‐induced liver injury in rats through an Akt‐dependent HO‐1 pathway.     

A catalytic di-heme bis-Fe(IV) intermediate, alternative to an Fe(IV)=O porphyrin radical

High-valent iron species are powerful oxidizing agents in chemical and biological catalysis. The best characterized form of an Fe(V) equivalent described in biological systems is the combination of a b-type heme with Fe(IV)=O and a porphyrin or amino acid cation radical (termed Compound I). This work describes an alternative natural mechanism to store two oxidizing equivalents above the ferric state for biological oxidation reactions. MauG is an enzyme that utilizes two covalently bound c-type hemes to catalyze the biosynthesis of the protein-derived cofactor tryptophan tryptophylquinone. Its natural substrate is a monohydroxylated tryptophan residue present in a 119-kDa precursor protein. An EPR-silent di-heme reaction intermediate of MauG was trapped. Mössbauer spectroscopy revealed the presence of two distinct Fe(IV) species. One is consistent with an Fe(IV)=O (ferryl) species (δ = 0.06 mm/s, ΔE_Q = 1.70 mm/s). The other is assigned to an Fe(IV) heme species with two axial ligands from protein (δ = 0.17 mm/s, ΔE_Q = 2.54 mm/s), which has never before been described in nature. This bis-Fe(IV) intermediate is remarkably stable but readily reacts with its native substrate. These findings broaden our views of how proteins can stabilize a highly reactive oxidizing species and the scope of enzyme-catalyzed posttranslational modifications.     

Hb F‐M‐Osaka [Gγ63(E7)His→Tyr] in a Newborn from Southwest France

We have monitored a sequence of reactions involving hemoglobin (Hb) oxidation in the presence of phospholipid bilayers in carefully characterized buffer systems in order to delineate molecular events that may have cytotoxic effects, some of which may even lead to cellular death of erythrocytes. We followed reactions of subunit cross-linking, heme iron oxidation, heme destruction and iron release in normal (Hb A) and sickle [Hb S or β6(A3)Glu→Val] Hbs under different experimental conditions. Our results show that, in the presence of lipid surfaces, the reaction rates for heme Fe^{+ 2} to Fe^{+ 3} oxidation in Hb A and Hb S molecules were both enhanced by lipid surfaces. However, the extent of the enhancement in Hb A and Hb S was quite different in T5K6.5, a Tris buffer with low ionic strength and low pH. In T5K6.5, the rate constants were 0.8 h^{? 1} for Hb A and 4.3 h^{? 1} for Hb S, a 5-fold difference. This finding supports a published suggestion that sickle Hb exhibits abnormal heme oxidation in erythrocytes of sickle cell disease patients. We found that the rates were quite similar in P110N7.4, a phosphate buffer near physiological conditions. Even in T5K6.5, the lipid surface did not enhance Hb S reactions involving subunit cross-linking, heme destruction, or iron release. These findings suggest that heme oxidation and related reactions may not be responsible for detrimental cellular events in sickle erythrocytes under physiological conditions, as suggested in the literature.     

Melatonin modulates neuroinflammation and oxidative stress in experimental diabetic neuropathy: effects on NF-κB and Nrf2 cascades

Abstract: Melatonin exhibits an array of biological activities, including antioxidant and anti‐inflammatory actions. Diabetic neuropathy is one of the complications of diabetes with a prevalence rate of 50–60%. We have previously reported the protective effect of melatonin in experimental diabetic neuropathy. In this study, we investigated the role of nuclear factor‐kappa B (NF‐κB) and nuclear erythroid 2‐related factor 2 (Nrf2) in melatonin‐mediated protection against streptozotocin‐induced diabetic neuropathy. Melatonin at doses of 3 and 10 mg/kg was administered daily in seventh and eighth week after diabetes induction. Motor nerve conduction velocity and nerve blood flow were improved in melatonin‐treated animals. Melatonin also reduced the elevated expression of NF‐κB, IκB‐α, and phosphorylated IκB‐α. Further, melatonin treatment also reduced the elevated levels of proinflammatory cytokines (TNF‐α and IL‐6), iNOS and COX‐2 in sciatic nerves of animals. The capacity of melatonin to modulate Nrf2 pathway was associated with increased heme oxygenase‐1 (HO‐1) expression, which strengthens antioxidant defense. This fact was also established by decreased DNA fragmentation (because inhibition of excessive oxidant‐induced DNA damage) in the sciatic nerve of melatonin‐treated animals. The results of this study suggest that melatonin modulates neuroinflammation by decreasing NF‐κB activation cascade and oxidative stress by increasing Nrf2 expression, which might be responsible at least in part, for its neuroprotective effect in diabetic neuropathy.     

Melatonin inhibits microglial activation, reduces pro-inflammatory cytokine levels, and rescues hippocampal neurons of adult rats with acute Klebsiella pneumoniae meningitis

Abstract: Acute bacterial meningitis caused by Klebsiella pneumoniae (K. pneumoniae) is a major health threat with a high mortality rate and severe neuro‐cognitive sequelae. The intense pro‐inflammatory cytokine released from calcium‐mediated microglial activation plays an important role in eliciting neuronal damage in the hippocampal region. Considering melatonin possesses anti‐inflammatory and immuno‐modulatory properties, the present study determined whether melatonin can effectively decrease inflammatory responses and prevent hippocampal damage in animals subjected to K. pneumoniae. Adult rats inoculated with K. pneumoniae received a melatonin injection immediately thereafter at doses of 5, 25, 50, or 100 mg/kg. Following 24 h of survival, all experimental animals were processed for time‐of‐flight secondary ion mass spectrometry (for detecting glial calcium intensity), isolectin‐B4 histochemistry (reliable marker for microglial activation), pro‐inflammatory cytokine measurement as well as cytochrome oxidase and in situ dUTP end‐labeling (representing neuronal bio‐energetic status and apoptotic changes, respectively). Results indicate that in K. pneumoniae‐infected rats, numerous calcium‐enriched microglia, enhanced pro‐inflammatory cytokine, and various apoptotic neurons with low bio‐energetic activity were detected in hippocampus. Following melatonin administration, however, all parameters including glial calcium intensity, microglial activation, pro‐inflammatory cytokine levels, and number of apoptotic neurons were successfully decreased with maximal change observed at a melatonin dose of 100 mg/kg. Enzymatic data corresponded well with above findings in which all surviving neurons displayed high bio‐energetic activity. As effectively reducing glia‐mediated inflammatory response is neuro‐protective to hippocampal neurons, the present study supports the clinical use of melatonin as a potential therapeutic agent to counteract K. pneumoniae meningitis‐induced neuro‐cognitive damage.     

Melatonin protects against experimental reflux esophagitis

Abstract:  Reflux esophagitis (RE), a major gastrointestinal disorder results from excess exposure of the esophageal mucosa to acidic gastric juice or bile-containing duodenal contents refluxed via an incompetent lower esophageal sphincter. Recent studies implicated oxygen derived free radicals in RE induced esophageal mucosal damage resulting in mucosal inflammation. Thus, control over free radical generation and modulation of inflammatory responses might offer better therapeutic effects to counteract the severity of RE. In this context we investigated the effect of melatonin against experimental RE in rats. Melatonin pretreatment significantly reduced the haemorrhagic lesions and decreased esophageal lipid peroxidation aggravated by RE. Moreover, the depleted levels of superoxide dismutase and glutathione observed in RE were replenished by melatonin signifying its free radical scavenging properties and antioxidant effects resulting in the improvement of esophageal defense mechanism. Further melatonin repressed the upregulated levels of expression of proinflammatory cytokines like, TNF-α, IL-1β and IL-6 in RE. However, increased levels of the anti-inflammatory cytokine IL-10 remained unaltered after melatonin administration signifying its immunomodulatory effect through suppression of Th1-mediated immune responses. The involvement of receptor dependent actions of melatonin against RE were also investigated with MT2 receptor antagonist, luzindole (LUZ). LUZ failed to antagonize melatonin's protective effects against RE indicating that melatonin mediated these beneficial effects in a receptor-independent fashion. Thus, esophageal mucosal protection elicited by melatonin against experimental RE is not only dependent on its free radical scavenging activity but also mediated in part through its effect on the associated inflammatory events in a receptor-independent manner.     

Intracellular kinetics of iron in reticulocytes: evidence for endosome involvement in iron targeting to mitochondria

In erythroid cells the vast majority of iron (Fe) released from endosomes must cross both the outer and the inner mitochondrial membranes to reach ferrochelatase that inserts Fe into protoporphyrin IX. In the present study, we developed a method whereby a cohort of 59Fe-transferrin (Tf)-laden endosomal vesicles were generated, from which we could evaluate the transfer of 59Fe into mitochondria. Iron chelators, dipyridyl or salicylaldehyde isonicotinoyl hydrazone (SIH), were able to bind the 59Fe when they were present during a 37 degrees C incubation; however, addition of these agents only during lysis at 4 degrees C chelated virtually no 59Fe. Bafilomycin A1 (which prevents endosome acidification) and succinylacetone (an inhibitor of 5-aminolevulinate dehydratase) prevented endosomal 59Fe incorporation into heme. Importantly, both the myosin light chain kinase inhibitor wortmannin and the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), caused significant inhibition of 59Fe incorporation from 59Fe-Tf-labeled endosomes into heme, suggesting that myosin is required for Tf-vesicle movement. Our results reaffirm the astonishing efficiency of Tf-derived Fe utilization in hemoglobin (Hb)-producing cells and demonstrate that very little of this Fe is present in a chelatable pool. Collectively, these results are congruent with our hypothesis that a transient endosome-mitochondrion interaction mediates iron transfer between these organelles.     

Melatonin reduces indomethacin‐induced gastric mucosal cell apoptosis by preventing mitochondrial oxidative stress and the activation of mitochondrial pathway of apoptosis

Abstract: Augmentation of gastric mucosal cell apoptosis due to development of oxidative stress is one of the main pathogenic events in the development of nonsteroidal anti‐inflammatory drug (NSAID)‐induced gastropathy. Identification of a nontoxic, anti‐apoptotic molecule is warranted for therapy against NSAID‐induced gastropathy. The objective of the present study was to define the mechanism of the anti‐apoptotic effect of melatonin, a nontoxic molecule which scavenges reactive oxygen species. Using an array of experimental approaches, we have shown that melatonin prevents the development of mitochondrial oxidative stress and activation of mitochondrial pathway of apoptosis induced by indomethacin (a NSAID) in the gastric mucosa. Melatonin inhibits the important steps of indomethacin‐induced activation of mitochondrial pathway of apoptosis such as upregulation of the expression of Bax and Bak, and the downregulation of Bcl‐2 and BclxL. Melatonin also prevents indomethacin‐induced mitochondrial translocation of Bax and prevents the collapse of mitochondrial membrane potential. Moreover, melatonin reduces indomethacin‐mediated activation of caspase‐9 and caspase‐3 by blocking the release of cytochrome c and finally rescues gastric mucosal cells from indomethacin‐induced apoptosis as measured by the TUNEL assay. Histologic studies of gastric mucosa further document that melatonin almost completely protects against gastric damage induced by indomethacin. Thus, melatonin has significant anti‐apoptotic effects to protect gastric mucosa from NSAID‐induced apoptosis and gastropathy, which makes its use as potential therapy against gastric damage during NSAID treatment.     

Melatonin protects bone marrow mesenchymal stem cells against iron overload‐induced aberrant differentiation and senescence

Bone marrow mesenchymal stem cells (BMSCs) are an expandable population of stem cells which can differentiate into osteoblasts, chondrocytes and adipocytes. Dysfunction of BMSCs in response to pathological stimuli contributes to bone diseases. Melatonin, a hormone secreted from pineal gland, has been proved to be an important mediator in bone formation and mineralization. The aim of this study was to investigate whether melatonin protected against iron overload‐induced dysfunction of BMSCs and its underlying mechanisms. Here, we found that iron overload induced by ferric ammonium citrate (FAC) caused irregularly morphological changes and markedly reduced the viability in BMSCs. Consistently, osteogenic differentiation of BMSCs was significantly inhibited by iron overload, but melatonin treatment rescued osteogenic differentiation of BMSCs. Furthermore, exposure to FAC led to the senescence in BMSCs, which was attenuated by melatonin as well. Meanwhile, melatonin was able to counter the reduction in cell proliferation by iron overload in BMSCs. In addition, protective effects of melatonin on iron overload‐induced dysfunction of BMSCs were abolished by its inhibitor luzindole. Also, melatonin protected BMSCs against iron overload‐induced ROS accumulation and membrane potential depolarization. Further study uncovered that melatonin inhibited the upregulation of p53, ERK and p38 protein expressions in BMSCs with iron overload. Collectively, melatonin plays a protective role in iron overload‐induced osteogenic differentiation dysfunction and senescence through blocking ROS accumulation and p53/ERK/p38 activation.     

Synergistic neuroprotective effect of combined low concentrations of galantamine and melatonin against oxidative stress in SH‐SY5Y neuroblastoma cells

Abstract: Melatonin is a potent free radical scavenger, antioxidant and neuroprotective drug. On the other hand, galantamine is a cholinergic drug with antioxidant and neuroprotective properties linked to inhibition of acetylcholinesterase and allosteric modulation of nicotinic receptors. This investigation evaluated a possible synergistic neuroprotective effect of subeffective concentrations of combined galantamine and melatonin. Human neuroblastoma SH‐SY5Y cells were subjected to a mitochondrial oxidative stress, by blockade of mitochondrial complexes I and V with rotenone and oligomycin‐A (R/O); cells were treated for 24 hr with R/O. This caused 40% of the cell to die as measured by lactate dehydrogenase (LDH) release. Cell incubation with increasing concentrations of galantamine (10–300 nm ) or melatonin (0.3–10 nm ) for 24 hr, followed by a 24‐hr period with R/O, caused a concentration‐dependent protection; maximum protection was achieved with 300 nm galantamine (56% protection) and 10 nm melatonin (50% protection). Combination of subeffective concentrations of melatonin (0.3 nm ) and galantamine (30 nm ) caused a synergistic and significant protection that was similar to the maximum protection afforded by effective concentrations of melatonin or galantamine alone. This protective effect was completely reversed when nicotinic and melatonin receptors were blocked respectively by mecamylamine and luzindole. The neuroprotective effect was prevented by chelerythrine, LY294002, and Sn (IV) protoporphyrin IX dichloride (SnPP), indicating the participation of the PKC/PI3K/Akt activation and induction of the antioxidant enzyme heme oxygenase‐1. The synthesis of novel multitarget compounds having in a single molecule the combined neuroprotective properties of galantamine and melatonin could be a new strategy for potential therapeutic agents in neurodegenerative diseases.     

The nature of heme/iron-induced protein tyrosine nitration

Recently, substantial evidence has emerged that revealed a very close association between the formation of nitrotyrosine and the presence of activated granulocytes containing peroxidases, such as myeloperoxidase. Peroxidases share heme-containing homology and can use H(2)O(2) to oxidize substrates. Heme is a complex of iron with protoporphyrin IX, and the iron-containing structure of heme has been shown to be an oxidant in several model systems where the prooxidant effects of free iron, heme, and hemoproteins may be attributed to the formation of hypervalent states of the heme iron. In the current study, we have tested the hypothesis that free heme and iron play a crucial role in NO(2)-Tyr formation. The data from our study indicate that: (i) hemeiron catalyzes nitration of tyrosine residues by using hydrogen peroxide and nitrite, a reaction that revealed the mechanism underlying the protein nitration by peroxidase, H(2)O(2), and NO(2)(-); (ii) H(2)O(2) plays a key role in the protein oxidation that forms the basis for the protein nitration, whereas nitrite is an essential element that facilitates nitration by the heme(Fe), H(2)O(2), and the NO(2)(-) system; (iii) the formation of a Fe(IV) hypervalent compound may be essential for heme(Fe)-catalyzed nitration, whereas O(2)(*-) (ONOO(-) formation), (*)OH (Fenton reaction), and compound III are unlikely to contribute to the reaction; and (iv) hemoprotein-rich tissues such as cardiac muscle are vulnerable to protein nitration in pathological conditions characterized by the overproduction of H(2)O(2) and NO(2)(-), or nitric oxide.     

Melatonin alleviates cadmium‐induced liver injury by inhibiting the TXNIP‐NLRP3 inflammasome

Cadmium (Cd) is a persistent environmental and occupational contaminant that accumulates in the liver and induces oxidative stress and inflammation. Melatonin possesses potent hepatoprotective properties against the development and progression of acute and chronic liver injury. Nevertheless, the molecular mechanism underlying the protective effects of melatonin against Cd‐induced hepatotoxicity remains obscure. In this study, we aimed to investigate the effects of melatonin on Cd‐induced liver inflammation and hepatocyte death. Male C57BL/6 mice were intraperitoneally injected with melatonin (10 mg/kg) once a day for 3 days before exposure to CdCl₂ (2.0 mg/kg). We found that Cd induced hepatocellular damage and inflammatory infiltration as well as increased serum ALT/AST enzymes. In addition, we showed that Cd triggered an inflammatory cell death, which is mediated by the NOD‐like receptor pyrin domain containing 3 (NLRP3) inflammasome. Moreover, melatonin treatment significantly alleviated Cd‐induced liver injury by decreasing serum ALT/AST levels, suppressing pro‐inflammatory cytokine production, inhibiting NLRP3 inflammasome activation, ameliorating oxidative stress, and attenuating hepatocyte death. Most importantly, melatonin markedly abrogated Cd‐induced TXNIP overexpression and decreased the interaction between TXNIP and NLRP3 in vivo and in vitro. However, treatment with siRNA targeting TXNIP blocked the protective effects of melatonin in Cd‐treated primary hepatocytes. Collectively, our results suggest that melatonin confers protection against Cd‐induced liver inflammation and hepatocyte death via inhibition of the TXNIP‐NLRP3 inflammasome pathway.     

Melatonin promotes angiogenesis during protection and healing of indomethacin‐induced gastric ulcer: role of matrix metaloproteinase‐2

Abstract: Matrix metalloproteinase (MMP)‐2 is considered as a crucial regulator of angiogenesis, a process of new blood vessel formation. We reported previously that melatonin (N‐acetyl‐5‐methoxy tryptamine), an antioxidant and anti‐inflammatory agent, prevents indomethacin‐induced gastric ulcers. Herein, we investigated the effect of melatonin on MMP‐2‐mediated angiogenesis during gastroprotection. Angiogenic properties of melatonin were tested in both rat corneal micropocket assay and in mouse model of indomethacin‐induced gastric lesions. Melatonin augmented angiogenesis that was associated with amelioration of MMP‐2 expression and activity and, upregulation of vascular endothelial growth factor (VEGF) in rat cornea. Melatonin prevented gastric lesions by promoting angiogenesis via upregulation of VEGF followed by over‐expression of MMP‐2. Similarly, healing of gastric lesions was associated with early expression of VEGF followed by MMP‐2. In addition, upregulation of MMP‐2 was parallel to MMP‐14 and inverse to tissue inhibitor of metalloprotease (TIMP)‐2 expression during gastroprotection. Our data demonstrated that melatonin exerts angiogenesis through MMP‐2 and VEGF over‐expression during protection and healing of gastric ulcers. This study highlights for the first time a phase‐associated regulation of MMP‐2 activity in gastric mucosa and an angiogenic action of melatonin to rescue indomethacin‐induced gastropathy.