A review of nitric oxide and oxidative stress in typical ovulatory women and in the pathogenesis of ovulatory dysfunction in PCOS.

Polycystic ovary syndrome (PCOS) is a heterogeneous functional endocrine disorder associated with a low-grade, chronic inflammatory state. Patients with PCOS present an increased risk of metabolic comorbidities and often menstrual dysregulation and infertility due to anovulation and/or poor oocyte quality. Multiple mechanisms including oxidative stress and low-grade inflammation are believed to be responsible for oocyte deterioration; however, the influence of nitric oxide (NO) insufficiency in oocyte quality and ovulatory dysfunction in PCOS is still a matter for debate. Higher production of superoxide (O2•-) mediated DNA damage and impaired antioxidant defense have been implicated as contributory factors for the development of PCOS, with reported alteration in superoxide dismutase (SOD) function, an imbalanced zinc/copper ratio, and increased catalase activity. These events may result in decreased hydrogen peroxide (H2O2) accumulation with increased lipid peroxidation events. A decrease in NO, potentially due to increased activity of NO synthase (NOS) inhibitors such as asymmetric dimethylarginine (ADMA), and imbalance in the distribution of reactive oxygen species (ROS), such as decreased H2O2 and increased O2•-, may offset the physiological processes surrounding follicular development, oocyte maturation, and ovulation contributing to the reproductive dysfunction in patients with PCOS. Thus, this proposal aims to evaluate the specific roles of NO, oxidative stress, ROS, and enzymatic and nonenzymatic elements in the pathogenesis of PCOS ovarian dysfunction, including oligo- anovulation and oocyte quality, with the intent to inspire better application of therapeutic options. The authors believe more consideration into the specific roles of oxidative stress, ROS, and enzymatic and nonenzymatic elements may allow for a more thorough understanding of PCOS. Future efforts elaborating on the role of NO in the preoptic nucleus to determine its influence on GnRH firing and follicle-stimulating hormone/Luteinizing hormone (FSH/LH) production with ovulation would be of benefit in PCOS. Consequently, treatment with an ADMA inhibitor or NO donor may prove beneficial to PCOS patients experiencing reproductive dysfunction and infertility.

Hypochlorous acid facilitates inducible nitric oxide synthase subunit dissociation: The link between heme destruction, disturbance of the zinc-tetrathiolate center, and the prevention by melatonin.

Inducible nitric oxide synthase (iNOS) is a zinc-containing hemoprotein composed of two identical subunits, each containing a reductase and an oxygenase domain. The reductase domain contains binding sites for NADPH, FAD, FMN, and tightly bound calmodulin and the oxygenase domain contains binding sites for heme, tetrahydrobiopterin (H4B), and l-arginine. The enzyme converts l-arginine into nitric oxide (NO) and citrulline in the presence of O2. It has previously been demonstrated that myeloperoxidase (MPO), which catalyzes formation of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride (Cl-), is enhanced in inflammatory diseases and could be a potent scavenger of NO. Using absorbance spectroscopy and gel filtration chromatography, we investigated the role of increasing concentrations of HOCl in mediating iNOS heme destruction and subsequent subunit dissociation and unfolding. The results showed that dimer iNOS dissociation between 15 and 100 µM HOCl was accompanied by loss of heme content and NO synthesis activity. The dissociated subunits-maintained cytochrome c and ferricyanide reductase activities. There was partial unfolding of the subunits at 300 µM HOCl and above, and the subunit unfolding transition was accompanied by loss of reductase activities. These events can be prevented when the enzyme is preincubated with melatonin prior to HOCl addition. Melatonin supplementation to patients experiencing low NO levels due to inflammatory diseases may be helpful to restore physiological NO functions.

Melatonin prevents hypochlorous acid-mediated cyanocobalamin destruction and cyanogen chloride generation.

Hypochlorous acid (HOCl) is a potent cytotoxic oxidant generated by the enzyme myeloperoxidase (MPO) in the presence of hydrogen peroxide (H2 O2 ) and chloride (Cl- ). Elevated levels of HOCl play an important role in various pathological conditions through oxidative modification of several biomolecules. Recently, we have highlighted the ability of HOCl to mediate the destruction of the metal-ion derivatives of tetrapyrrole macrocyclic rings such as hemoproteins and vitamin B12 (VB12 ) derivatives. Destruction of cyanocobalamin, a common pharmacological form of VB12 mediated by HOCl, 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 (CNCl), and redox-active free cobalt. Here, we show that melatonin prevents HOCl-mediated cyanocobalamin destruction, using a combination of UV-Vis spectrophotometry, high-performance liquid chromatography analysis, and colorimetric CNCl assay. Identification of several melatonin oxidation products suggests that the protective role of melatonin against HOCl-mediated cyanocobalamin destruction and subsequent CNCl 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 VB12 deficiency in inflammatory conditions such as cardiovascular and neurodegenerative diseases, among many others.

Computational analysis of nitric oxide biotransport to red blood cell in the presence of free hemoglobin and NO donor.

Red blood cells (RBCs) modulate nitric oxide (NO) bioavailability in the vasculature. Extracellular free hemoglobin (Hb) in the vascular lumen can cause NO bioavailability related complications seen in pathological conditions such as pancreatitis, sickle cell disease and malaria. In addition, the role of extracellular free Hb has been critical to estimate kinetic and transport properties of NO-RBCs interactions in 'competition experiments'. We recently reported a strong dependence of NO transport on RBC membrane permeability and hematocrit. NO donors combined with anti-inflammatory drugs are an emergent treatment for diseases like cancer, cardiovascular complications and wound healing. However, the role of RBCs in transport NO from NO donors is not clearly understood. To understand the significance of extracellular free Hb in pathophysiology on NO availability and estimation of the NO-RBC interactions, we developed a computational model to simulate NO biotransport to the RBC in the presence of extracellular free Hb. Using this model, we studied the effect of hematocrit, RBC membrane permeability and NO donors on NO-RBC interactions in the presence and absence of extracellular free Hb. The plasma NO concentration gradients and average plasma NO concentrations changed minimally with increase in extracellular free Hb concentrations at the higher hematocrit as compared to those at the lower hematocrit irrespective of the NO delivery method, indicating that the presence of extracellular free Hb affects the NO transport only at a low hematocrit. We also observed that NO concentrations increased with NO donor concentrations in the absence as well as in the presence of extracellular free Hb. In addition, NO donor supplementation may increase NO availability in the plasma in the event of loss of endothelium-derived NO activity.

The Implications of Insufficient Zinc on the Generation of Oxidative Stress Leading to Decreased Oocyte Quality.

Zinc is a transition metal that displays wide physiological implications ranging from participation in hundreds of enzymes and proteins to normal growth and development. In the reproductive tract of both sexes, zinc maintains a functional role in spermatogenesis, ovulation, fertilization, normal pregnancy, fetal development, and parturition. In this work, we review evidence to date regarding the importance of zinc in oocyte maturation and development, with emphasis on the role of key zinc-binding proteins, as well as examine the effects of zinc and reactive oxygen species (ROS) on oocyte quality and female fertility. We summarize our current knowledge about the participation of zinc in the developing oocyte bound to zinc finger proteins as well as loosely bound zinc ion in the intracellular and extracellular environments. These include aspects related to (1) the impact of zinc deficiency and overwhelming production of ROS under inflammatory conditions on the offset of the physiological antioxidant machinery disturbing biomolecules, proteins, and cellular processes, and their role in contributing to further oxidative stress; (2) the role of ROS in modulating damage to proteins containing zinc, such as zinc finger proteins and nitric oxide synthases (NOS), and expelling the zinc resulting in loss of protein function; and (3) clarify the different role of oxidative stress and zinc deficiency in the pathophysiology of infertility diseases with special emphasis on endometriosis-associated infertility.

Diminishing oocyte quality with advancing age is associated with deficiency of nitric oxide synthase cofactors, tetrahydrobiopterin, and zinc, in mouse oocytes.

OBJECTIVE: To study the implications of decreased zinc and tetrahydrobiopterin (H4B) associated with chronological aging on oocyte quality using a mouse model. H4B and zinc are essential cofactors for nitric oxide synthase (NOS), because they aid in electron transfer and dimeric stability, and their bioavailability is crucial in regulating NOS coupling. We have previously shown that sufficient levels of nitric oxide (NO) are essential for maintaining oocyte quality and activation, and NO levels decrease in the oocyte as a function of age. Thus, it is plausible that zinc and H4B may decrease as a function of age, resulting in NOS dysfunction with subsequent depletion of NO. Additionally, increased production of reactive oxygen species from the monomeric form can further disrupt oocyte quality and NO bioavailability. DESIGN: Experimental laboratory study. SETTING: Laboratory. ANIMALS: B6D2F1 mice. INTERVENTION(S): Sibling oocytes were retrieved from super-ovulated B6D2F1 mice from 3 age groups: 8-14 weeks (young breeders [YBs]), 48-52 weeks (retired breeders [RBs]), and 80-84 weeks (old animals [OAs]). MAIN OUTCOME MEASURE(S): Oocytes were scored for ooplasmic/spindle microtubule (MT) morphology, chromosomal alignment, and cortical granule (CG) intactness using immunofluorescence and confocal microscopy with 3 dimension image reconstruction and subjected to an high-performance liquid chromatography assay to measure the concentrations of H4B and its metabolites, as well as the zinc measurement using atomic absorption spectrophotometry. RESULT(S): Oocyte scoring showed a reduction in "good" quality oocyte percentage as age increases, with YB having the highest percentage of quality oocytes followed by RB and OA. The high-performance liquid chromatography analysis showed a significant progressive decrease in total H4B in RB and OA (0.00098 picogram (pg)/oocyte and 0.00069 pg/oocyte, respectively) compared with YB (0.00125 pg/oocyte). Atomic absorbance spectrophotometry revealed a significant progressive decrease in zinc concentration in RB and OA compared with YB (8.45 pg/oocyte and 5.82 pg/oocyte vs. 10.05 pg/oocyte, respectively). CONCLUSION(S): Age-related diminution in oocyte quality is paralleled by a decline in the levels of H4B and zinc. The resultant deficiency in the oocytes can lead to the inability of NOS to maintain dimerization. Consequent uncoupling of NOS generates superoxide instead of NO, which participates in a multitude of reactions contributing to oxidative stress. Therefore, dysfunction of NOS secondary to zinc and H4B loss is a major mechanism involved in reactive oxygen species generation and oocyte quality deterioration related to the chronological age.

Determinants of Embryo Implantation: Roles of the Endometrium and Embryo in Implantation Success.

Both uterine endometrium and embryo contribute to implantation success. However, their relative role in the implantation success is still a matter for debate, as are the roles of endometrial receptivity analysis (ERA), endometrial scratch (ES), endometrial microbiome, and intrauterine or intravenous measures that are currently advocated to improve the implantation success. There is insufficient evidence to suggest that the endometrium is more important than the embryo in determining the implantation success and the utility of these measures, especially when euploid embryos are transferred is limited. Although embryo implantation on epithelium other than the endometrium is a very rare event, evidence suggests that embryo implantation and growth is not limited to the endometrium alone. Embryos can implant and develop to result in livebirths on epithelium that lacks the typical endometrial development present at implantation. Currently, the role of embryo euploidy in implantation success is underappreciated. At a minimum, it is the author's opinion that until robust, definitive studies are conducted that demonstrate benefit, reproductive endocrinologists and infertility specialist should be prudent in the way they counsel patients about the utility of ERA, ES, and other measures in improving implantation success.

Chronological age enhances aging phenomena and protein nitration in oocyte.

Background: The average age of childbearing has increased over the years contributing to infertility, miscarriages, and chromosomal abnormalities largely invoked by an age-related decline in oocyte quality. In this study, we investigate the role of nitric oxide (NO) insufficiency and protein nitration in oocyte chronological aging. Methods: Mouse oocytes were retrieved from young breeders (YB, 8-14 weeks [w]), retired breeders (RB, 48-52w) and old animals (OA, 80-84w) at 13.5 and 17 hours after ovulation trigger. They were assessed for zona pellucida dissolution time (ZPDT); ooplasmic microtubule dynamics (OMD); cortical granule (CG) status and spindle morphology (SM), as markers of oocyte quality. Sibling oocytes from RB were exposed to NO supplementation and assessed for aging phenomena (AP). All oocyte cumulus complexes were subjected to fluorescence nitrotyrosine (NT) immunocytochemistry and confocal microscopy to assess morphology and protein nitration. Results: At 13.5 h from hCG trigger, oocytes from RB compared to YB had significantly increased ZPDT (37.8 ± 11.9 vs 22.1 ± 4.1 seconds [s]), OMD (46.9 vs 0%), CG loss (39.4 vs 0%), and decreased normal SM (30.3 vs 81.3%), indicating premature AP that worsened among oocytes from RB at 17 hours post-hCG trigger. When exposed to SNAP, RB AP significantly decreased (ZPDT: 35.1 ± 5.5 vs 46.3 ± 8.9s, OMD: 13.3 vs 75.0% and CG loss: 50.0 vs 93.3%) and SM improved (80.0 vs 14.3%). The incidence of NT positivity was significantly higher in cumulus cells (13.5 h, 46.7 ± 4.5 vs 3.4 ± 0.7%; 17 h, 82.2 ± 2.9 vs 23.3 ± 3.6%) and oocytes (13.5 h, 57.1 vs 0%; 17 h, 100.0 vs 55.5%) from RB compared to YB. Oocytes retrieved decreased with advancing age (29.8 ± 4.1 per animal in the YB group compared to 10.2 ± 2.1 in RB and 4.0 ± 1.6 in OA). Oocytes from OA displayed increased ZPDT, major CG loss, increased OMD and spindle abnormalities, as well as pronuclear formation, confirming spontaneous meiosis to interphase transition. Conclusions: Oocytes undergo zona pellucida hardening, altered spindle and ooplasmic microtubules, and premature cortical granule release, indicative of spontaneous meiosis-interphase transition, as a function of chronological aging. These changes are also associated with NO insufficiency and protein nitration and may be alleviated through supplementation with an NO-donor.

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

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, 200nmol/mL), HOCl (10, 20, 50, and 100nmol/mL), and HOCl (50nmol/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 50nmol/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 50nmol/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.

Melatonin attenuates hypochlorous acid-mediated heme destruction, free iron release, and protein aggregation in hemoglobin.

In inflammatory diseases, where hypochlorous acid (HOCl) is elevated, iron homeostasis is disturbed, resulting in accumulation of free iron. Free iron is toxic by virtue of its ability to generate free radicals through the Fenton reaction. HOCl is generated by myeloperoxidase, (MPO) using chloride and hydrogen peroxide as substrates. Recent studies demonstrate that HOCl binds to the heme moiety of hemoglobin (Hb), which generates a transient ferric species whose formation and decay kinetics indicate it participates in protein aggregation, heme destruction, and free iron release. Here, we show that melatonin prevents HOCl-mediated Hb heme destruction and protein aggregation, using a combination of UV-vis spectrophotometry, ferrozine colorimetric assay, and in-gel heme staining. We also show that melatonin treatment prevents HOCl-mediated loss of red blood cell (RBC) viability, indicating biologic relevance of this finding. The mechanism by which melatonin prevents HOCl-mediated Hb heme destruction is by direct scavenging of HOCl and/or through the destabilization of the higher Hb oxidative states intermediates, ferryl porphyrin radical cation Hb-Fe(IV)=O(+π•) and Hb-Fe(IV)=O, which are formed through the reaction of HOCl with Hb. Our work establishes a direct mechanistic link between melatonin and its protective effect in chronic inflammatory diseases. Collectively, in addition to acting as an antioxidant and as a MPO inhibitor, melatonin can also exert its protective effect by inhibiting HOCl-mediated heme destruction of hemoproteins and subsequent free iron release.

Potential Role of Zinc in the COVID-19 Disease Process and its Probable Impact on Reproduction.

COVID-19 (coronavirus disease 2019) is the current world health crisis, producing extensive morbidity and mortality across all age groups. Given the established roles of zinc in combating oxidative damage and viral infections, zinc is being trialed as a treatment modality against COVID-19. Zinc also has confirmed roles in both male and female reproduction. The possible depletion of zinc with the oxidative events of COVID-19 is especially relevant to the fertility of affected couples. This review aims to present the pathophysiology of COVID-19, especially in relation to reproductive function; the role of zinc in the COVID-19 disease process; and how zinc depletion in concert with cytokine storm and reactive oxygen species production could affect reproduction. It also highlights research areas to better the understanding of COVID-19 and its impact on fertility and potential ways to mitigate the impact.

Modulation of redox signaling promotes apoptosis in epithelial ovarian cancer cells.

OBJECTIVE: Epithelial ovarian cancer (EOC) cells are known to be resistant to apoptosis through a mechanism that may involve alteration in their redox balance. NADPH oxidase is a major source of intracellular superoxide, which is converted to the less toxic product by superoxide dismutase (SOD). Superoxide contributes to hypoxia inducible factor (HIF)-1α stabilization. We sought to determine the effects of inhibiting the generation of intracellular reactive oxygen species (ROS) on apoptosis of EOC cells. METHODS: Diphenyleneiodonium (DPI), an irreversible ROS inhibitor, was used to inhibit the generation of ROS in EOC cell lines, SKOV-3 and MDAH-2774, followed by assessment of apoptosis, NADPH oxidase, SOD3 and HIF-1α expression. A combination of immunohistochemistry, immunoprecipitation/western blot, and real-time RT-PCR were utilized to evaluate the expression of these enzymes in EOC cells as well as normal ovarian tissue and ovarian cancer tissue specimens. RESULTS: DPI treatment significantly induced apoptosis in both EOC cell lines as evident by increased caspase-3 activity and TUNEL assay. Additionally, both EOC cell lines were found to express NADPH oxidase, HIF-1α, and SOD3, which were highly sensitive to DPI treatment. DPI treatment resulted in reduced NADPH oxidase, SOD3 and HIF-1α levels. Furthermore, ovarian cancer tissues were found to manifest higher NADPH oxidase levels as compared to normal ovarian tissues. CONCLUSIONS: These data suggest that lowering oxidative stress, possibly through the inhibition of NADPH oxidase, induces apoptosis in ovarian cancer cells and may serve as a potential target for cancer therapy.

A Multiple-Hit Hypothesis Involving Reactive Oxygen Species and Myeloperoxidase Explains Clinical Deterioration and Fatality in COVID-19.

Multi-system involvement and rapid clinical deterioration are hallmarks of coronavirus disease 2019 (COVID-19) related mortality. The unique clinical phenomena in severe COVID-19 can be perplexing, and they include disproportionately severe hypoxemia relative to lung alveolar-parenchymal pathology and rapid clinical deterioration, with poor response to O2 supplementation, despite preserved lung mechanics. Factors such as microvascular injury, thromboembolism, pulmonary hypertension, and alteration in hemoglobin structure and function could play important roles. Overwhelming immune response associated with "cytokine storms" could activate reactive oxygen species (ROS), which may result in consumption of nitric oxide (NO), a critical vasodilation regulator. In other inflammatory infections, activated neutrophils are known to release myeloperoxidase (MPO) in a natural immune response, which contributes to production of hypochlorous acid (HOCl). However, during overwhelming inflammation, HOCl competes with O2 at heme binding sites, decreasing O2 saturation. Moreover, HOCl contributes to several oxidative reactions, including hemoglobin-heme iron oxidation, heme destruction, and subsequent release of free iron, which mediates toxic tissue injury through additional generation of ROS and NO consumption. Connecting these reactions in a multi-hit model can explain generalized tissue damage, vasoconstriction, severe hypoxia, and precipitous clinical deterioration in critically ill COVID-19 patients. Understanding these mechanisms is critical to develop therapeutic strategies to combat COVID-19.

Melatonin interferes with COVID-19 at several distinct ROS-related steps.

Recent studies have shown a correlation between COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and the distinct, exaggerated immune response titled "cytokine storm". This immune response leads to excessive production and accumulation of reactive oxygen species (ROS) that cause clinical signs characteristic of COVID-19 such as decreased oxygen saturation, alteration of hemoglobin properties, decreased nitric oxide (NO) bioavailability, vasoconstriction, elevated cytokines, cardiac and/or renal injury, enhanced D-dimer, leukocytosis, and an increased neutrophil to lymphocyte ratio. Particularly, neutrophil myeloperoxidase (MPO) is thought to be especially abundant and, as a result, contributes substantially to oxidative stress and the pathophysiology of COVID-19. Conversely, melatonin, a potent MPO inhibitor, has been noted for its anti-inflammatory, anti-oxidative, anti-apoptotic, and neuroprotective actions. Melatonin has been proposed as a safe therapeutic agent for COVID-19 recently, having been given with a US Food and Drug Administration emergency authorized cocktail, REGEN-COV2, for management of COVID-19 progression. This review distinctly highlights both how the destructive interactions of HOCl with tetrapyrrole rings may contribute to oxygen deficiency and hypoxia, vitamin B12 deficiency, NO deficiency, increased oxidative stress, and sleep disturbance, as well as how melatonin acts to prevent these events, thereby improving COVID-19 prognosis.

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.

Hypochlorous acid reversibly inhibits caspase-3: a potential regulator of apoptosis.

Caspase-3 is involved in apoptosis. Here, we examine whether hypochlorous acid (HOCl), a final product of myeloperoxidase (MPO), is a modulator of caspase-3 at relatively low concentrations and also its application on metaphase II mouse oocytes. We utilised caspase-3 activity assay, TUNEL assay, the CellEvent caspase 3/7 fluorescent assay, and the MPO/hydrogen peroxide (H2O2) system on mouse oocytes with and without cumulus cells to examine whether low concentrations of HOCl mediate apoptosis by inhibition of caspase-3. A UV-visible spectrophotometer was used to study caspase-3 activity. To determine whether HOCl mediates apoptosis in mouse oocytes, two different concentrations (10 and 100 µM) of HOCl generated by the MPO/H2O2 system were used as treatments (10 µM had little effect on oocyte quality, while 100 µM showed significant deterioration). Induction of apoptotic cell death was determined by TUNEL Assay and the CellEvent caspase 3/7. HOCl mediates caspase-3 inactivation in a dose dependent manner. Subsequent addition of dithiothreitol caused recovery of caspase-3 activity indicating involvement of the oxidation of the Cys-thiol group. Accumulation of HOCl generated by MPO in the presence of caspase-3 also inhibits MPO but requires higher HOCl concentrations, indicating specificity of lower HOCl concentrations to inhibition of caspase-3. Exposure of oocytes to lower HOCl concentrations generated by MPO-H2O2 system prevents MPO-mediated apoptosis whereas exposure to higher HOCl (100 µM) showed apoptosis. Similar results were observed by using the CellEvent caspase 3/7 assay. Low concentrations of HOCl inhibit caspase-3 activity, and may play a role in regulating apoptosis, thus affecting oocyte quality.HighlightsCaspase-3 is involved in apoptosis pathway and loss of this regulation is seen in several diseases.These conditions are associated with inflammation and higher myeloperoxidase (MPO) activity.We examined whether hypochlorous acid (HOCl), generated by MPO, is a modulator of caspase-3.Caspase-3 activity showed a dose dependent decrease with HOCl and this reaction was reversible.HOCl modulates caspase-3 activity and may play a physiological role in regulating apoptosis.

Glyphosate Induces Metaphase II Oocyte Deterioration and Embryo Damage by Zinc Depletion and Overproduction of Reactive Oxygen Species.

Glyphosate is the most popular herbicide used in modern agriculture, and its use has been increasing substantially since its introduction. Accordingly, glyphosate exposure from food and water, the environment, and accidental and occupational venues has also increased. Recent studies have demonstrated a relationship between glyphosate exposure and a number of disorders such as cancer, immune and metabolic disorders, endocrine disruption, imbalance of intestinal flora, cardiovascular disease, and infertility; these results have given glyphosate a considerable amount of media and scientific attention. Notably, glyphosate is a powerful metal chelator, which could help explain some of its effects. Recently, our findings on 2,3-dimercapto-1-propanesulfonic acid, another metal chelator, showed deterioration of oocyte quality. Here, to generalize, we investigated the effects of glyphosate (0 - 300 µM) on metaphase II mouse oocyte quality and embryo damage to obtain insight on its mechanisms of cellular action and the tolerance of oocytes and embryos towards this chemical. Our work shows for the first time that glyphosate exposure impairs metaphase II mouse oocyte quality via two mechanisms: 1) disruption of the microtubule organizing center and chromosomes such as anomalous pericentrin formation, spindle fiber destruction and disappearance, and defective chromosomal alignment and 2) substantial depletion of intracellular zinc bioavailability and enhancement of reactive oxygen species accumulation. Similar effects were found in embryos. These results may help clarify the effects of glyphosate exposure on female fertility and provide counseling and preventative steps for excessive glyphosate intake and resulting oxidative stress and reduced zinc bioavailability.

The inhibition of lactoperoxidase catalytic activity through mesna (2-mercaptoethane sodium sulfonate).

Here, we show that mesna (sodium-2-mercaptoethane sulfonate), primarily used to prevent nephrotoxicity and urinary tract toxicity caused by chemotherapeutic agents such as cyclophosphamide and ifosfamide, modulates the catalytic activity of lactoperoxidase (LPO) by binding tightly to the enzyme, functioning either as a one electron substrate for LPO Compounds I and II, destabilizing Compound III. Lactoperoxidase is a hemoprotein that utilizes hydrogen peroxide (H2O2) and thiocyanate (SCN-) to produce hypothiocyanous acid (HOSCN), an antimicrobial agent also thought to be associated with carcinogenesis. Our results revealed that mesna binds stably to LPO within the SCN- binding site, dependent of the heme iron moiety, and its combination with LPO-Fe(III) is associated with a disturbance in the water molecule network in the heme cavity. At low concentrations, mesna accelerated the formation and decay of LPO compound II via its ability to serve as a one electron substrate for LPO compounds I and II. At higher concentrations, mesna also accelerated the formation of Compound II but it decays to LPO-Fe(III) directly or through the formation of an intermediate, Compound I*, that displays characteristic spectrum similar to that of LPO Compound I. Mesna inhibits LPO's halogenation activity (IC50 value of 9.08 µM) by switching the reaction from a 2e- to a 1e- pathway, allowing the enzyme to function with significant peroxidase activity (conversion of H2O2 to H2O without generation of HOSCN). Collectively, mesna interaction with LPO may serve as a potential mechanism for modulating its steady-state catalysis, impacting the regulation of local inflammatory and infectious events.

The role of myeloperoxidase in the pathogenesis of postoperative adhesions.

Hypoxia induces the adhesion phenotype, characterized by enhanced extracellular matrix molecule and cytokine expression. Additionally, hypoxia reduces myeloperoxidase (MPO) activity in normal peritoneal fibroblasts to basal levels of adhesion fibroblasts indicating the importance of this enzyme in the development of the adhesion phenotype and also in tissue fibrosis. Immunohistochemistry was used to detect and localize MPO and inducible nitric oxide synthase (iNOS) in fibroblasts. Silencing of these genes was performed using siRNA technology. Levels of iNOS, MPO, type I collagen, and transforming growth factor were detected using real-time reverse transcription-polymerase chain reaction (RT-PCR), while HPLC was used to measure nitrate/nitrite levels. Our results show a unique interaction between MPO and iNOS, which are colocalized in both cell lines. Silencing iNOS reduced MPO and nitric oxide levels while silencing MPO had similar results, but to a lesser extent in both cell types. Additionally, silencing iNOS reduced type I collagen and transforming growth factor-beta in adhesion fibroblasts, but to a lesser extent in peritoneal fibroblasts. These studies identify MPO and iNOS as key enzymes in the cellular response to hypoxia and consequent development of tissue fibrosis.

S-nitrosylation of caspase-3 is the mechanism by which adhesion fibroblasts manifest lower apoptosis.

We have previously found that adhesion fibroblasts exhibit lower apoptosis and higher protein nitration as compared with normal peritoneal fibroblasts. In this study, we sought to determine whether the decreased apoptosis observed in adhesion fibroblasts is caused by lower caspase-3 activity due to an increase in caspase-3 S-nitrosylation. For this study, we have utilized primary cultures of fibroblasts obtained from normal peritoneum and adhesion tissues of the same patient(s). Cells were treated with increasing concentrations of peroxynitrite and cell lysates were immunoprecipitated with anti-caspase-3 polyclonal antibody. The biotinylated proteins were detected using a nitrosylation detection kit. Caspase-3 activity and apoptosis were measured by colorimetric and TUNEL assays, respectively. Our results showed that caspase-3 S-nitrosylation is significantly higher in adhesion fibroblasts as compared with normal peritoneal fibroblasts. This increase in S-nitrosylation resulted in a 30% decrease in caspase-3 activity in adhesion fibroblasts. Peroxynitrite treatment resulted in a dose response increase in caspase-3 S-nitrosylation, leading to a decrease in caspase-3 activity and apoptosis in normal peritoneal fibroblasts. We conclude that S-nitrosylation of caspase-3 is the reason for its decreased activity and subsequent decrease in apoptosis of adhesion fibroblasts. The mechanism by which caspase-3 S-nitrosylation occurs is not fully understood. However, the role of hypoxia in the formation of peroxynitrite via superoxide production may suggest a possible mechanism.