Husam M. Abu-Soud

Melatonin Is a Potent Inhibitor for Myeloperoxidase

Myeloperoxidase (MPO) catalyzes the formation of potent oxidants that have been implicated in the pathogenesis of various diseases including atherosclerosis, asthma, arthritis, and cancer. Melatonin plays an important part in the regulation of various body functions including circadian sleep rhythms, blood pressure, oncogenesis, retinal function, seasonal reproduction, and immunity. Here, we demonstrate that melatonin serves as a potent inhibitor of MPO under physiological-like conditions. In the presence of chloride (Cl-), melatonin inactivated MPO at two points in the classic peroxidase cycle through binding to MPO to form an inactive complex, melatonin-MPO-Cl, and accelerating MPO compound II formation, an inactive form of MPO. Inactivation of MPO was mirrored by the direct conversion of MPO-Fe(III) to MPO compound II without any sign of compound I accumulation. This behavior indicates that melatonin binding modulates the formation of MPO intermediates and their decay rates. The Cl- presence enhanced the affinity of MPO toward melatonin, which switches the enzyme activity from peroxidation to catalase-like activity. In the absence of Cl-, melatonin served as a 1e- substrate for MPO compound I, but at higher concentration it limited the reaction by its dissociation from the corresponding complex. Importantly, melatonin-dependent inhibition of MPO occurred with a wide range of concentrations that span various physiological and supplemental ranges. Thus, the interplay between MPO and melatonin may have a broader implication in the function of several biological systems. This dual regulation by melatonin is unique and represents a new means through which melatonin can control MPO and its downstream inflammatory pathways.

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.

Myeloperoxidase up-regulates the catalytic activity of inducible nitric oxide synthase by preventing nitric oxide feedback inhibition

Kinetic and structure analysis of inducible nitric oxide synthase (iNOS) revealed that, in addition to the increase of iNOS expression in inflamed areas, the major pathway causing overproduction of NO is destabilization of the iNOS–nitrosyl complex(es) that form during steady-state catalysis. Formation of such a complex allows iNOS to operate at only a fraction (20–30%) of its maximum activity. Thus, bioavailability of NO scavengers at sites of inflammation may play an essential role in up-regulation of the catalytic activity of iNOS, by preventing the catalytic activity inhibition that is attributed to nitrosyl complex formation. Myeloperoxidase (MPO), a major NO scavenger, is a pivotal enzyme involved in leukocyte-mediated host defenses. It is thought to play a pathogenic role under circumstances such as acute inflammatory tissue injury and chronic inflammatory conditions. However, a detailed understanding of the interrelationship between iNOS and MPO at sites of inflammation is lacking. We used direct spectroscopic, HPLC, and selective NO-electrode measurements to determine the interdependent relationship that exists between iNOS and MPO and the role of the MPO/H2O2 system in up-regulating the catalytic activity of iNOS that occurs at sites of inflammation. Scavenging free NO from the iNOS milieu by the MPO/H2O2 system subsequently restores the full capacity of iNOS to convert l-aginine to product (NO), as judged by the increase in the rates of citrulline and nitrite/nitrate production. Studies of iNOS catalytic mechanisms and function are essential to a more fundamental understanding of these factors, which govern iNOS-dependent processes in human health and disease.

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.

Hypoxia-generated superoxide induces the development of the adhesion phenotype

Adhesion fibroblasts exhibit higher TGF-beta1 and type I collagen expression as compared to normal peritoneal fibroblasts. Furthermore, exposure of normal peritoneal fibroblasts to hypoxia results in an irreversible increase in TGF-beta1 and type I collagen. We postulated that the mechanism by which hypoxia induced the adhesion phenotype is through the production of superoxide either directly or through the formation of peroxynitrite. To test this hypothesis, normal peritoneal and adhesion fibroblasts were treated with superoxide dismutase (SOD), a superoxide scavenger, and xanthine/xanthine oxidase, a superoxide-generating system, under normoxic and hypoxic conditions. Also, cells were treated with peroxynitrite. TGF-beta1 and type I collagen expression was determined before and after all treatments using real-time RT/PCR. Hypoxia treatment resulted in a time-dependent increase in TGF-beta1 and type I collagen mRNA levels in both normal peritoneal and adhesion fibroblasts. Similarly, treatment with xanthine oxidase, to endogenously generate superoxide, resulted in higher mRNA levels of TGF-beta1 and type I collagen in both normal peritoneal and adhesion fibroblasts. In contrast, treatment with SOD, to scavenge endogenous superoxide, resulted in a decrease in TGF-beta1 and type I collagen expression in adhesion fibroblasts to levels seen in normal peritoneal fibroblasts; no effect on the expression of these molecules was seen in normal peritoneal fibroblasts. Exposure to hypoxia in the presence of SOD had no effect on mRNA levels of TGF-beta1 and type I collagen in either normal peritoneal or adhesion fibroblasts. Peroxynitrite treatment alone significantly induced both adhesion phenotype markers. In conclusion, hypoxia, through the production of superoxide, causes normal peritoneal fibroblasts to acquire the adhesion phenotype. Scavenging superoxide, even in the presence of hypoxia, prevented the development of the adhesion phenotype. These findings further support the central role of free radicals in the development of adhesions.

Nitric oxide extends the oocyte temporal window for optimal fertilization.

Deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age. In this study, we explore the role of nitric oxide (NO) on the sustenance of oocyte quality postovulation. Sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (ICSI) with cauda-epididymal spermatozoa following exposure to either the NO donor, S-nitroso-N-acetylpenicillamine (SNAP, 0.23 microM/min), an NO synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 1 mM), or an inhibitor of soluble guanylyl cyclase (sGC), 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, 100 microM); while their sibling oocytes were subjected to ICSI either before (young) or after culture for the corresponding period of time (old). Outcomes of normal fertilization, cleavage, and development to the morula and blastocyst stages were compared. Embryos from each subgroup were also subjected to TUNEL assay for apoptosis. A significant deterioration in the ability of the oocytes to undergo normal fertilization and development to morula and blastocyst stages occurred among oocytes aged in culture medium compared to their sibling cohorts subjected to ICSI immediately after ovulation (P<0.05). This deterioration was prevented in oocytes exposed to SNAP. In contrast, exposure to L-NAME or ODQ resulted in a significant compromise in fertilization and development to the morula and blastocyst stages (P<0.05). Finally, apoptosis was noted in embryos derived from aged oocytes and those exposed to L-NAME or ODQ, but not in embryos derived from young oocytes or oocytes exposed to SNAP. Thus, NO is essential for sustenance of oocyte quality postovulation.

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.

Dichloroacetate induces apoptosis of epithelial ovarian cancer cells through a mechanism involving modulation of oxidative stress.

Epithelial ovarian cancer (EOC) cells are under intrinsic oxidative stress, which alters metabolic activity and reduces apoptosis. Key oxidative stress enzymes, including myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS), are upregulated and colocalized in EOC cells. Oxidative stress is also regulated, in part, by superoxide dismutase (SOD) and hypoxia-inducible factor (HIF) 1a. Dichloroacetate (DCA) converts anaerobic to aerobic metabolism and thus was utilized to determine the effects on apoptosis, iNOS, MPO, extracellular SOD (SOD-3), and HIF-1a, in EOC cells. Protein and messenger RNA (mRNA) levels of iNOS, MPO, SOD-3, and HIF-1a were evaluated by immunoprecipitation/Western blot and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively, utilizing SKOV-3 and MDAH-2774 treated with DCA. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3 assays. Dichloroacetate induced apoptosis, reduced MPO, iNOS, and HIF-1a, whereas increased SOD, in both EOC cell lines. In conclusion, reduction of enhanced oxidative stress-induced apoptosis of EOC cells, which may serve as future therapeutic intervention for ovarian cancer.

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.

Myeloperoxidase Serves as a Redox Switch that Regulates Apoptosis in Epithelial Ovarian Cancer

OBJECTIVES Resistance to apoptosis is a key feature of cancer cells and is believed to be regulated by nitrosonium ion (NO(+))-induced S-nitrosylation of key enzymes. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is utilized by MPO to generated NO(+). We sought to investigate the expression of myeloperoxidase (MPO) and iNOS in epithelial ovarian cancer (EOC) and determine their effect on S-nitrosylation of caspase-3 and its activity as well as apoptosis. METHODS MPO and iNOS expression were determined using immunofluorescence in SKOV-3 and MDAH-2774 and EOC tissue sections. S-nitrosylation of caspase-3 and its activity, levels of MPO and iNOS, as well as apoptosis, were evaluated in the EOC cells before and after silencing MPO or iNOS genes with specific siRNA probes utilizing real-time RT-PCR, ELISA, and TUNEL assays. RESULTS MPO and iNOS are expressed in EOC cell lines and in over 60% of invasive EOC cases with no expression in normal ovarian epithelium. Indeed, silencing of MPO or iNOS gene expression resulted in decreased S-nitrosylation of caspase-3, increased caspase-3 activity, and increased apoptosis but with a more significant effect when silencing MPO. CONCLUSION MPO and iNOS are colocalized to the same cells in EOC but not in the normal ovarian epithelium. Silencing of either MPO or iNOS significantly induced apoptosis, highlighting their role as a redox switch that regulates apoptosis in EOC. Understanding the mechanisms by which MPO functions as a redox switch in regulating apoptosis in EOC may lead to future diagnostic tools and therapeutic interventions.