Electrochemical Reduction of Perfluorooctanoic Acid (PFOA): An Experimental and Theoretical Approach.

Perfluorooctanoic acid (PFOA) is an artificial chemical of global concern due to its high environmental persistence and potential human health risk. Electrochemical methods are promising technologies for water treatment because they are efficient, cheap, and scalable. The electrochemical reduction of PFOA is one of the current methodologies. This process leads to defluorination of the carbon chain to hydrogenated products. Here, we describe a mechanistic study of the electrochemical reduction of PFOA in gold electrodes. By using linear sweep voltammetry (LSV), an E0' of -1.80 V vs Ag/AgCl was estimated. Using a scan rate diagnosis, we determined an electron-transfer coefficient (αexp) of 0.37, corresponding to a concerted mechanism. The strong adsorption of PFOA into the gold surface is confirmed by the Langmuir-like isotherm in the absence (KA = 1.89 × 1012 cm3 mol-1) and presence of a negative potential (KA = 3.94 × 107 cm3 mol-1, at -1.40 V vs Ag/AgCl). Based on Marcus-Hush's theory, calculations show a solvent reorganization energy (λ0) of 0.9 eV, suggesting a large electrostatic repulsion between the perfluorinated chain and water. The estimated free energy of the transition state of the electron transfer (ΔG‡ = 2.42 eV) suggests that it is thermodynamically the reaction-limiting step. 19F - 1H NMR, UV-vis, and mass spectrometry studies confirm the displacement of fluorine atoms by hydrogen. Density functional theory (DFT) calculations also support the concerted mechanism for the reductive defluorination of PFOA, in agreement with the experimental values.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Catalase prevents myeloperoxidase self-destruction in response to oxidative stress.

Catalase (CAT) and myeloperoxiase (MPO) are heme-containing enzymes that have attracted attention for their role in the etiology of numerous respiratory disorders such as cystic fibrosis, bronchial asthma, and acute hypoxemic respiratory failure. However, information regarding the interrelationship and competition between the two enzymes, free iron accumulation, and decreased levels of non-enzymatic antioxidants at sites of inflammation is still lacking. Myeloperoxidase catalyzes the generation of hypochlorous acid (HOCl) from the reaction of hydrogen peroxide (H2O2) and chloride (Cl-). Self-generated HOCl has recently been proposed to auto-inhibit MPO through a mechanism that involves MPO heme destruction. Here, we investigate the interplay of MPO, HOCl, and CAT during catalysis, and explore the crucial role of MPO inhibitors and HOCl scavengers in protecting the catalytic site from protein modification of both enzymes against oxidative damage mediated by HOCl. We showed that CAT not only competes with MPO for H2O2 but also scavenges HOCl. The protective role provided by CAT versus the damaging effect provided by HOCl depends in part on the ratio between MPO/CAT and the affinity of the enzymes towards H2O2 versus HOCl. The severity of such damaging effects mainly depends on the ratio of HOCl to enzyme heme content. In addition to its effect in mediating protein modification and aggregation, HOCl oxidatively destroys the catalytic sites of the enzymes, which contain porphyrin rings and iron. Thus, modulation of MPO/CAT activities may be a fundamental feature of catalysis, and functions to down-regulate HOCl synthesis and prevent hemoprotein heme destruction and/or protein modification.

Mesenchymal deficiency of Notch1 attenuates bleomycin-induced pulmonary fibrosis.

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct in vivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)-bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I-expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.