Dhiman Maitra

Potent Antioxidative Activity of Lycopene: A Potential Role in Scavenging Hypochlorous Acid

Lycopene, a carotenoid found in tomatoes, is a proven antioxidant that may lower the risk of certain disorders including heart disease and cancer. Hypochlorous acid (HOCl) is an oxidant linked to tissue oxidation in cardiovascular disease and other inflammatory disorders through its ability to modify proteins, deoxyribonucleic acid, ribonucleic acid, and lipids. Here we show that lycopene can function as a potent scavenger of HOCl at a wide range of concentrations that span various pathophysiological and supplemental ranges. The oxidation of lycopene by HOCl was accompanied by a marked change in color, from red to colorless, of the lycopene solution, suggesting lycopene degradation. HPLC and LC-MS analysis showed that the exposure of lycopene to increasing concentrations of HOCl gave a range of metabolites resulting from oxidative cleavage of one or more C=C. The degree of degradation of lycopene (as assessed by the number and chain lengths of the various oxidative metabolites of lycopene) depends mainly on the ratio of HOCl to lycopene, suggesting that multiple molecules of HOCl are consumed per molecule of lycopene. Collectively, this work demonstrates a direct link between lycopene and HOCl scavenging and may assist in elucidating the mechanism of the protective function exerted by lycopene.

Reaction of hemoglobin with HOCl: Mechanism of heme destruction and free iron release

Hypochlorous acid (HOCl) is generated by myeloperoxidase using chloride and hydrogen peroxide as substrates. HOCl and its conjugate base (OCl(-)) bind to the heme moiety of hemoglobin (Hb) and generate a transient ferric species whose formation and decay kinetics indicate it can participate in protein aggregation and heme destruction along with subsequent free iron release. The oxidation of the Hb heme moiety by OCl(-) was accompanied by marked heme destruction as judged by the decrease in and subsequent flattening of the Soret absorbance peak at 405 nm. HOCl-mediated Hb heme depletion was confirmed by HPLC analysis and in-gel heme staining. Exposure of Hb to increasing concentrations of HOCl produced a number of porphyrin degradation products resulting from oxidative cleavage of one or more of the carbon-methene bridges of the tetrapyrrole ring, as identified by their characteristic HPLC fluorescence and LC-MS. A nonreducing denaturing SDS-PAGE showed several degrees of protein aggregation. Similarly, porphyrin degradation products were identified after exposure of red blood cells to increasing concentrations of HOCl, indicating biological relevance of this finding. This work provides a direct link between Hb heme destruction and subsequent free iron accumulation, as occurs under inflammatory conditions where HOCl is formed in substantial amounts.

Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway

Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl(-)) and hydrogen peroxide (H₂O₂). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV-visible spectra and H₂O₂-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H₂O₂ (10 µM), precluding the enzyme from functioning at maximum activity (80-90% inhibition). MPO heme destruction occurred only in the presence of Cl(-). Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO-Compound I-Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron.

Mechanism of hypochlorous acid mediated heme destruction and free iron release

Here, we show that hypochlorous acid (HOCl), a potent neutrophil-generated oxidant, can mediate destruction of free heme (Ht) and the heme precursor, protoporphyrin IX (PPIX). Ht displays a broad Soret absorbance peak centered at 365 and 394 nm, indicative of the presence of monomer and μ-oxo-dimer. Oxidation of Ht by HOCl was accompanied by a marked decrease in the Soret absorption peak and release of free iron. Kinetic measurements showed that the Ht-HOCl reaction was triphasic. The first two phases were HOCl concentration dependent and attributable to HOCl binding to the monomeric and dimeric forms. The third phase was HOCl concentration independent and attributed to Ht destruction with the release of free iron. HPLC and LC-ESI-MS analyses of the Ht-HOCl reaction revealed the formation of a number of degradation products, resulting from the cleavage or modification of one or more carbon-methene bridges of the porphyrin ring. Similar studies with PPIX showed that HOCl also mediated tetrapyrrole ring destruction. Collectively, this work demonstrates the ability of HOCl to modulate destruction of heme, through a process that occurs independent of the iron molecule that resides in the porphyrin center. This phenomenon may play a role in HOCl-mediated oxidative injury in pathological conditions.

Melatonin prevents hypochlorous acid‐induced alterations in microtubule and chromosomal structure in metaphase‐II mouse oocytes

Abstract:  Hypochlorous acid (HOCl) is generated by myeloperoxidase, using chloride and hydrogen peroxide as substrates. Here we demonstrate that HOCl alters metaphase‐II mouse oocyte microtubules and chromosomal (CH) alignment which can be prevented by melatonin. Metaphase‐II mouse oocytes, obtained commercially, were grouped as: control, melatonin (150, 200 nmol/mL), HOCl (10, 20, 50, and 100 nmol/mL), and HOCl (50 nmol/mL) pretreated with 150 and 200 nmol/mL of melatonin. Microtubule and CH alignment was studied utilizing an indirect immunofluorescence technique and scored by two observers. Pearson chi‐square test and Fisher’s exact test were used to compare outcomes between controls and treated groups and also among each group. Poor scores for the spindle and chromosomes increased significantly at 50 nmol/mL of HOCl (P < 0.001). Oocytes treated with melatonin only at 150 and 200 nmol/mL showed no changes; significant differences (P < 0.001) were observed when oocytes exposed to 50 nmol/mL of HOCl were compared to oocytes pretreated with 200 nmol/mL melatonin. Fifty percent of the oocytes demonstrated good scores, both in microtubule and CH alterations, when pretreated with melatonin at 150 nmol/mL compared to 0% in the HOCl‐only group. HOCl alters the metaphase‐II mouse oocyte spindle and CH alignment in a dose‐dependant manner, which might be a potential cause of poor oocyte quality (e.g., in patients with endometriosis). Melatonin prevented the HOCl‐mediated spindle and CH damage, and therefore, may be an attractive therapeutic option to prevent oocyte damage in endometriosis or inflammatory diseases where HOCl levels are known to be elevated.

Myeloperoxidase interaction with peroxynitrite: chloride deficiency and heme depletion

Myeloperoxidase (MPO) is a hemoprotein involved in the leukocyte-mediated defense mechanism and uses hydrogen peroxide (H2O2) and chloride (Cl(-)) to produce hypochlorous acid. In human saliva and in hypochloremic alkalosis syndrome occurring in breast-fed infants, the MPO-H2O2 system functions in a lower Cl(-) concentration (10-70 mM) compared to plasma levels (100 mM) as part of the antibacterial defense system. The impact of low Cl(-) concentration and exposure to high peroxynitrite (ONOO(-)) synthesized from cigarette smoke or oxidative stress on MPO function is still unexplored. Rapid mixing of ONOO(-) and MPO caused immediate formation of a transient intermediate MPO Compound II, which then decayed to MPO-Fe(III). Double mixing of MPO with ONOO(-) followed by H2O2 caused immediate formation of Compound II, followed by MPO heme depletion, a process that occurred independent of ONOO(-) concentration. Peroxynitrite/H2O2-mediated MPO heme depletion was confirmed by HPLC analysis, and in-gel heme staining showing 60-70% less heme content compared to the control. A nonreducing denaturing SDS-PAGE showed no fragmentation or degradation of protein. Myeloperoxidase heme loss was completely prevented by preincubation of MPO with saturating amounts of Cl(-). Chloride binding to the active site of MPO constrains ONOO(-) binding by filling the space directly above the heme moiety or by causing a protein conformational change that constricts the distal heme pocket, thus preventing ONOO(-) from binding to MPO heme iron. Peroxynitrite interaction with MPO may serve as a novel mechanism for modulating MPO catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.

Hypochlorous Acid-Induced Heme Degradation from Lactoperoxidase as a Novel Mechanism of Free Iron Release and Tissue Injury in Inflammatory Diseases

Lactoperoxidase (LPO) is the major consumer of hydrogen peroxide (H2O2) in the airways through its ability to oxidize thiocyanate (SCN−) to produce hypothiocyanous acid, an antimicrobial agent. In nasal inflammatory diseases, such as cystic fibrosis, both LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by neutrophils, are known to co-localize. The aim of this study was to assess the interaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Our rapid kinetic measurements revealed that HOCl binds rapidly and reversibly to LPO-Fe(III) to form the LPO-Fe(III)-OCl complex, which in turn decayed irreversibly to LPO Compound II through the formation of Compound I. The decay rate constant of Compound II decreased with increasing HOCl concentration with an inflection point at 100 µM HOCl, after which the decay rate increased. This point of inflection is the critical concentration of HOCl beyond which HOCl switches its role, from mediating destabilization of LPO Compound II to LPO heme destruction. Lactoperoxidase heme destruction was associated with protein aggregation, free iron release, and formation of a number of fluorescent heme degradation products. Similar results were obtained when LPO-Fe(II)-O2, Compound III, was exposed to HOCl. Heme destruction can be partially or completely prevented in the presence of SCN−. On the basis of the present results we concluded that a complex bi-directional relationship exists between LPO activity and HOCl levels at sites of inflammation; LPO serve as a catalytic sink for HOCl, while HOCl serves to modulate LPO catalytic activity, bioavailability, and function.

The reaction of HOCl and cyanocobalamin: Corrin destruction and the liberation of cyanogen chloride

Overproduction of hypochlorous acid (HOCl) has been associated with the development of a variety of disorders such as inflammation, heart disease, pulmonary fibrosis, and cancer through its ability to modify various biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl and heme destruction and free iron release from hemoglobin and RBCs. Our current findings extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl), a common pharmacological form of vitamin B12. Cyanocobalamin is a water-soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that although Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC, and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphonucleotide moiety. Additionally, it can liberate free Co, which can perpetuate metal-ion-induced oxidant stress. Taken together, these results are the first report of the generation of toxic molecular products through the interaction of Cobl with HOCl.

Impact of hydrogen peroxide driven Fenton reaction on mouse oocyte quality

Here we show that hydroxyl radical ((•)OH) generated through the Fenton reaction alters metaphase-II mouse oocyte microtubules (MT) and chromosomal alignment (CH). Metaphase-II mouse oocytes, obtained commercially, were grouped as follows: control, hydrogen peroxide (H2O2), Fe(II), and combined (Fe(II) +H2O2) treatments. After 7-10 min of incubation at 37 °C, MT and CH were evaluated on fixed and stained oocytes and scored by two blinded observers. Pearson χ(2) test and Fisher exact test were used to compare outcomes between controls and treated groups and also among the treated groups. Our results showed that poor scores for MT and CH increased significantly in oocytes treated with a combination of H2O2 and Fe(II) (p<0.001); oocytes treated with H2O2 alone or Fe(II) alone showed no or few changes compared to control. Comparison of oocyte groups that received increasing concentrations of H2O2 and a fixed amount of Fe(II) showed that 70-80% demonstrated poor scores in both MT and CH when pretreated with 5 μM H2O2, and this increased up to 90-100% when treated with 10-20 μM H2O2. Hydroxyl radical generated by H2O2-driven Fenton reaction deteriorates the metaphase-II mouse oocyte spindle and CH alignment, which is thought to be a potential cause of poor oocyte quality. Thus, free iron and/or ROS scavengers could attenuate the (•)OH-mediated spindle and chromosomal damage, thereby serving as a possible approach for further examination as a therapeutic option in inflammatory states.

IL-6 and Mouse Oocyte Spindle

Interleukin 6 (IL-6) is considered a major indicator of the acute-phase inflammatory response. Endometriosis and pelvic inflammation, diseases that manifest elevated levels of IL-6, are commonly associated with higher infertility. However, the mechanistic link between elevated levels of IL-6 and poor oocyte quality is still unclear. In this work, we explored the direct role of this cytokine as a possible mediator for impaired oocyte spindle and chromosomal structure, which is a critical hurdle in the management of infertility. Metaphase-II mouse oocytes were exposed to recombinant mouse IL-6 (50, 100 and 200 ng/mL) for 30 minutes and subjected to indirect immunofluorescent staining to identify alterations in the microtubule and chromosomal alignment compared to untreated controls. The deterioration in microtubule and chromosomal alignment were evaluated utilizing both fluorescence and confocal microscopy, and were quantitated with a previously reported scoring system. Our results showed that IL-6 caused a dose-dependent deterioration in microtubule and chromosomal alignment in the treated oocytes as compared to the untreated group. Indeed, IL-6 at a concentration as low as 50 ng/mL caused deterioration in the spindle structure in 60% of the oocytes, which increased significantly (P<0.0001) as IL-6 concentration was increased. In conclusion, elevated levels of IL-6 associated with endometriosis and pelvic inflammation may reduce the fertilizing capacity of human oocyte through a mechanism that involves impairment of the microtubule and chromosomal structure.