Effects of ambient ozone exposure on circulating extracellular vehicle microRNA levels in coronary artery disease patients.

Exposure to ambient air pollutants such as ozone (O3) and particulate matter (PM) is associated with increased cardiovascular morbidity and rate of mortality, but the underlying biological mechanisms have yet to be described. Emerging evidence shows that extracellular vehicle (EV) microRNAs (miRNAs) may facilitate cell-to-cell and organ-to-organ communications and play a role in the air pollution-induced cardiovascular effects. This study aims to explore the association between air pollutant exposure and miRNA changes related to cardiovascular diseases. Using a panel study design, 14 participants with coronary artery diseases were enrolled in this study. Each participant had up to 10 clinical visits and their plasma samples were collected and measured for expression of miRNA-21 (miR-21), miR-126, miR-146, miR-150, and miR-155. Mixed effects models adjusted for temperature, humidity, and season were used to examine the association between miRNA levels and exposure to 8-hr O3 or 24-hr PM2.5 up to 4 days prior. Results demonstrated that miR-150 expression was positively associated with O3 exposure at 1-4 days lag and 5day moving average while miR-155 expression tracked with O3 exposure at lag 0. No significant association was found between miRNA expression and ambient PM2.5 at any time point. ß-blocker and diabetic medication usage significantly modified the correlation between O3 exposure and miR-150 expression where the link was more prominent among non-users. In conclusion, evidence indicated an association between exposure to ambient O3 and circulating levels of EV miR-150 and miR-155 was observed. These findings pointed to a future research direction involving miRNA-mediated mechanisms of O3-induced cardiovascular effects.

Ozone Exposure Increases Circulating Stress Hormones and Lipid Metabolites in Humans.

RATIONALE: Air pollution has been associated with increased prevalence of type 2 diabetes; however, the mechanisms remain unknown. We have shown that acute ozone exposure in rats induces release of stress hormones, hyperglycemia, leptinemia, and glucose intolerance that are associated with global changes in peripheral glucose, lipid, and amino acid metabolism. OBJECTIVES: To examine ozone-induced metabolic derangement in humans using serum metabolomic assessment, establish human-to-rodent coherence, and identify novel nonprotein biomarkers. METHODS: Serum samples were obtained from a crossover clinical study that included two clinic visits (n = 24 each) where each subject was blindly exposed in the morning to either filtered air or 0.3 parts per million ozone for 2 hours during 15-minute on-off exercise. Serum samples collected within 1 hour after exposure were assessed for changes in metabolites using a metabolomic approach. MEASUREMENTS AND MAIN RESULTS: Metabolomic analysis revealed that ozone exposure markedly increased serum cortisol and corticosterone together with increases in monoacylglycerol, glycerol, and medium- and long-chain free fatty acids, reflective of lipid mobilization and catabolism. Additionally, ozone exposure increased serum lysolipids, potentially originating from membrane lipid breakdown. Ozone exposure also increased circulating mitochondrial ß-oxidation-derived metabolites, such as acylcarnitines, together with increases in the ketone body 3-hydroxybutyrate. These changes suggested saturation of ß-oxidation by ozone in exercising humans. CONCLUSIONS: As in rodents, acute ozone exposure increased stress hormones and globally altered peripheral lipid metabolism in humans, likely through activation of a neurohormonally mediated stress response pathway. The metabolomic assessment revealed new biomarkers and allowed for establishment of rodent-to-human coherence. Clinical trial registered with www.clinicaltrials.gov (NCT 01492517).

Combination of LC-MS- and GC-MS-based metabolomics to study the effect of ozonated autohemotherapy on human blood.

Ozonated autohemotherapy (O3-AHT) is a medical approach during which blood obtained from the patient is ozonated and injected back into the body. Despite an increasing number of evidence that O3-AHT is safe, this type of therapy remains controversial. To extend knowledge about the changes in blood evoked by O3-AHT, LC-MS- and GC-MS-based metabolic fingerprinting was used to compare plasma samples obtained from blood before and after the treatment with potentially therapeutic concentrations of ozone. The procedure was performed in PVC bags utilized for blood storage to study also possible interactions between ozone and plastic. By use of GC-MS, an increase in lactic acid and pyruvic acid was observed, which indicated an increased rate of glycolysis. With LC-MS, changes in plasma antioxidants were observed. Moreover, concentrations of lipid oxidation products (LOP) and lysophospholipids were increased after ozone treatment. This is the first report of increased LOPs metabolites after ozonation of blood. Seven metabolites detected by LC-QTOF-MS only in ozonated samples could be considered as novel biomarkers of oxidative stress. Several plasticizers have been detected by both techniques in blood stored in PVC bags. PVC is known to be an ozone resistant material, but ozonation of blood in PVC bags stimulates leaching of plasticizers into the blood.

CORonavirus-19 mild to moderate pneumonia Management with blood Ozonization in patients with Respiratory failure (CORMOR) multicentric prospective randomized clinical trial.

BACKGROUND: Following positive experience on the use of blood ozonation in SARS-CoV-2, the CORMOR randomized trial was designed to evaluate the adjuvant role of oxygen/ozone therapy in mild to moderate SARS-CoV-2 pneumonia. METHODS: The trial (ClinicalTrial.gov NCT04388514) was conducted in four different Italian centers (April-October 2020). Patients were treated according to best available standard of care (SoC) therapy, with or without O3-autohemotherapy (O3-AHT). RESULTS: A total of 92 patients were enrolled: SoC + O3-AHT (48 patients) were compared to the SoC treatment (44 patients). The two groups differed in steroids therapy administration (72.7% in SoC arm vs. 50.0% in O3-AHT arm; p = 0.044). Steroid therapy was routinely started when it was subsequently deemed as effective for the treatment of COVID-19 disease. No significant differences in mortality rates, length of hospital stay, mechanical ventilation requirement and ICU admission were observed. Clinical improvement in patients with pneumonia was assessed according to a specifically designed score (decrease in SIMEU class, improvement in radiology imaging, improvement in PaO2/FiO2, reduction in LDH and requirement of oxygen therapy ≤ 5 days). Score assessment was performed on day-3 (T3) and day-7 (TEnd) of O3-AHT treatment. A significant increase in the score was reported at TEnd, in the O3-AHT treatment arm (0 [0-1] in the SoC arm vs. 2 [1-3] the O3-AHT arm; p = 0.018). No adverse events related O3-AHT treatment was observed. CONCLUSION: In mild-to-moderate pneumonia due to SARS-CoV-2, adjuvant oxygen/ozone therapy did not show any effect on mortality, or mechanical intubation but show a clinical improvement a day 7 from randomization in a composite clinical endpoint. Larger Randomized prospective studies alone or in combination with steroids are needed to confirm our results.

Ozone: A Multifaceted Molecule with Unexpected Therapeutic Activity.

A comprehensive outline for understanding and recommending the therapeutic use of ozone in combination with established therapy in diseases characterized by a chronic oxidative stress is currently available. The view of the absolute ozone toxicity is incorrect, because it has been based either on lung or on studies performed in artificial environments that do not correspond to the real antioxidant capacity of body compartments. In fact, ozone exerts either a potent toxic activity or it can stimulate biological responses of vital importance, analogously to gases with prospective therapeutic value such as NO, CO, H2S, H2, as well as O2 itself. Such a crucial difference has increasingly become evident during the last decade. The purpose of this review is to explain the aspects still poorly understood, highlighting the divergent activity of ozone on the various biological districts. It will be clarified that such a dual effect does not depend only upon the final gas concentration, but also on the particular biological system where ozone acts. The real significance of ozone as adjuvant therapeutic treatment concerns severe chronic pathologies among which are cardiovascular diseases, chronic obstructive pulmonary diseases, multiple sclerosis, and the dry form of age-related macular degeneration. It is time for a full insertion of ozone therapy within pharmaceutical sciences, responding to all the requirements of quality, efficacy and safety, rather than as either an alternative or an esoteric approach.

Effects of semicarbazide on oxidative processes in human red blood cell membranes.

Semicarbazide can interfere with oxidative processes in the red blood cell membrane via different modes of action. Treatment of human red blood cell membranes with O3, results, among other effects, in cross-linking of membrane proteins and inhibition of glyceraldehyde-3-phosphate dehydrogenase activity. Semicarbazide inhibits these effects by acting as an O3 scavenger. The effect of semicarbazide as an O3 scavenger is complicated by the fact that ozonolysis of semicarbazide yields a product that causes inhibition of glyceraldehyde-3-phosphate dehydrogenase. Glyceraldehyde-3-phosphate dehydrogenase inhibition can also be provoked by incubation of membrane suspensions with O3-treated phospholipids. Semicarbazide prevented this effect by interaction with an inhibitory O3-phospholipid reaction product. Protoporphyrin-induced photodynamic cross-linking of membrane proteins is chemically distinct from O3-induced cross-linking. Photodynamic cross-linking is also inhibited by semicarbazide, in this case via reaction with a histidine photooxidation product.

Validity of Oxygen-Ozone Therapy as Integrated Medication Form in Chronic Inflammatory Diseases.

The state-of-the-art of oxygen-ozone therapy is now clarified and all the mechanisms of action of medical ozone are within classical biochemistry and molecular biology. The outcomes of standard treatments in peripheral arterial occlusive disease (PAOD) and dry-form of age-related macular degeneration (AMD) have been compared with the documented therapeutic results achieved with ozonated autohemotherapy (O-AHT). On the other hand, the clinical data of O-AHT on stroke remain indicative. As the cost of O-AHT is almost irrelevant, its application in all public hospitals, especially those of poor Countries, would allow two advantages: the first is for the patient, who will improve her/his conditions, and the second is for Health Authorities burdened with increasing costs. The aim of this paper is to report to clinical scientists that O-AHT is a scientific-based therapeutic approach without side effects. The integration of O-AHT with effective approved drugs is likely to yield the best clinical results in several chronic inflammatory diseases.

Oxidative damage to plasma constituents by ozone.

The reaction of ozone (O3) with human blood plasma was studied to help understand possible events that could occur in the respiratory tract. Uric acid (quantitatively the most important scavenger) and ascorbic acid were oxidized quickly, protein-SH groups were lost more slowly, and there was no loss of bilirubin or alpha-tocopherol. There was little formation of lipid hydroperoxides and no detectable formation of 4-hydroxynoneal, hexanal or nonanal, or changes in lipoprotein electrophoretic mobility. Uric acid in human upper airway secretions may play a significant role in removing inhaled O3. Oxidative damage to lipids must not be assumed to be the key mechanism of respiratory tract O3 toxicity.

Combined exposure to ozone and nitrogen dioxide.

The combined effects of ozone and nitrogen dioxide were assessed in an in vitro test system utilizing human red cells. In general, these two pollutants had additive effects on the parameters measured which included osmotic fragility, acetylcholinesterase activity, lipid peroxidation, reduced glutathione and methemoglobin levels. However, at lower pollutant doses a synergistic increase in lipid peroxides was noted while at higher doses the effect became less than additive. Further studies of this observation suggested that ferrous hemoglobin potentiates ozone-induced lipid peroxidation while methemoglobin, resulting primarily from nitrogen dioxide, inhibits this process. Ozone was also found to potentiate the methemoglobinemic effects of nitrogen dioxide, particularly in sequential studies in which ozone exposure preceded nitrogen dioxide. Inasmuch as the effects of these two pollutants vary from protective to synergistic depending on the pollutant concentration, duration and sequence of exposure, as well as on the parameter assayed, it would appear that the approach to in vivo study of the combined effects of ozone and nitrogen dioxide should be aimed at simulating ambient conditions as closely as possible.

Effect of O(2)/O(3) atmosphere on the rate of osmotic hemolysis of bovine erythrocytes in the presence of some antioxidants.

Purified red blood cells, exposed to an ozone atmosphere, show an increased rate of hemolysis on sudden osmotic stress. To determine this effect of ozone in the presence of natural antioxidants, bovine red blood cells, used as models, were suspended in blood plasma, or in physiological saline with one of the following antioxidants: albumin, glutathione, uric acid, glucose and a vitamin E analog (trolox). After exposure of the suspensions to oxygen and oxygen/ozone atmospheres the rates of osmotic hemolysis were measured, using a stop-flow technique, and compared with rates measured in air-exposed controls. Blood plasma, containing all natural antioxidants, caused a decreased rate of osmotic hemolysis of cells exposed to oxygen and also decreased the effect of ozone. Trolox cancelled the oxygen effects only. Albumin, glutathione and uric acid tended to protect the cells from the hemolytic effects of ozone. The antihemolytic effect of glucose, seen only in some samples, may depend on uncontrolled factors. The alteration of the rates correlates with an increased fluidity of red cell membranes exposed to ozone.

Ozonation of blood during extracorporeal circulation. I. Rationale, methodology and preliminary studies.

We investigated whether exposure of blood ex-vivo to oxygen-ozone (O2-O3) through a gas exchanger is feasible and practical. We first evaluated the classical dialysis-type technique but we soon realized that semipermeable membranes are unsuitable because they are hydrophilic and vulnerable to O3. We therefore adopted a system with hydrophobic O3-resistant hollow fibers enclosed in a polycarbonate housing with a membrane area of about 0.5 m2. First we tested the system with normal saline, determining the production of hydrogen peroxide (H2O2) at O3 concentrations from 5 to 40 microg/ml. We then evaluated critical parameters by circulating swine blood in vitro; this revealed that heparin is not an ideal anticoagulant for this system. Finally, we performed several experiments in sheep and defined optimal anticoagulant dose (sodium citrate, ACD), priming solution, volume of blood flow per min, volume and concentration of O2-O3 mixture flowing countercurrent with respect to blood and the time necessary for perfusion in vivo. The biochemical parameters showed that an O3 concentration as low as 10 microg/ml is effective; this means that gas exchange and O3 reactivity are rapid and capable of inducing biological effects. The sheep showed no adverse effects even after 50 min of extracorporeal circulation at higher O3 concentrations (20 to 40 microg/ml) but the exchanger became less effective (low pO2 values) due to progressive clogging with cells.

Ozone and human blood.

Statistically significant changes (P less than .05) were observed in erythrocytes (RBC) and sera of young adult human males following a single short-term exposure to 0.50 ppm ozone (O3) for 2 3/4 hours. The RBC membrane fragility, glucose-6-phosphate dehydrogenase (G-6-PDH) and lactate dehydrogenase (LDH) enzyme activities were increased, while RBC acetylcholinesterase (AcChase) activity and reduced glutathione (GSH) levels were decreased. The RBC glutathione reductase (GSSRase) activities were not significantly altered. Serum GSSRase activity, however, was significantly decreased while serum vitamin E, and lipid peroxidation levels were significantly increased. These alterations tend to disappear gradually, but were still detectable two weeks following exposure.

Free radical aspects of the ozone influence on blood.

Ozone is one of the most potent toxic agents. The noxious effects of its influence on living organisms appear mainly in the lungs and blood. A considerable role in its action is ascribed to free radicals. In this study the ESR method was applied to indirectly demonstrate the free radical effects, as well as the significance of ascorbate in ozonizing the blood. The effects of ozonizing the blood in vitro indicate the existence of a "local" protective mechanism of the blood cells, while in vivo they point out the consumption of ascorbate in "scavenging" of free radicals.

[Preliminary report on using general ozone therapy in diseases of the posterior segment of the eye]. / Wstepne doniesienie o stosowaniu ozonoterapii ogólnej w schorzeniach tylnego bieguna galki ocznej.

Autotransfusion of ozone venous blood was performed in 174 patients, in 32 patients ozone was given intraarterially. General ++ozone therapy was used for the first time in Poland in the following ophthalmological conditions: retinal pigment dystrophy, glaucomatous optic atrophy, in optic neuritis, after injuries and in cases of unknown aetiology; in degenerative-atrophic changes of the choroid, in high myopia, in post-inflammatory cases, in other degenerative diseases and in bacterial corneal ulcerations. Ozone therapy in these conditions seems to be favourable especially when the pathological process is not extensive. One could observe an improvement of the visual acuity and of the visual field.

An integrated medical treatment for type-2 diabetes.

This paper tries to emphasize two relevant concepts: the first is that type 2 diabetes is a chronic diseases characterized by both a dysmetabolism and a chronic oxidative stress. A variety of orthodox drugs are somewhat able to correct the metabolic alterations, but do not deal with the chronic inflammation. Consequently, as the validity of precisely treating blood with therapeutic ozone concentrations in restoring a redox homeostasis has been now demonstrated, the integration of ozone therapy appears essential for a rational treatment of type 2 diabetes. Such a combination may be able to reduce the diabetic epidemic.

How a calculated oxidative stress can yield multiple therapeutic effects.

It is proposed to discuss how ozonetherapy acts on patients affected by vascular and degenerative diseases. Ozone is a strong oxidant but, if used in small dosages on human blood ex vivo, acts as an acceptable stressor. By instantly reacting with PUFA bound to albumin, ozone is entirely consumed but generates two messengers acting in an early and in a late phase: the former is due to hydrogen peroxide, which triggers biochemical pathways on blood cells and the latter is due to alkenals which are infused into the donor patient. After undergoing a partial catabolism, alkenals enter into a great number of body's cells, where they react with Nrf2-Keap1 protein: the transfer of activated Nrf2 into the nucleus and its binding to antioxidant response element (ARE) is the crucial event able to upregulate the synthesis of antioxidant proteins, phase II enzymes and HO-1. With the progress of ozonetherapy, these protective enzymes are able to reverse the oxidative stress induced by chronic inflammation. Consequently, the repetition of graduated stresses induces a multiform adaptive response able to block the progress of the disease and to improve the quality of life.

Randomised, double-blinded, placebo-controlled, clinical trial of ozone therapy as treatment of sudden sensorineural hearing loss.

OBJECTIVE: To investigate the safety and efficacy of ozone therapy in adult patients with sudden sensorineural hearing loss. DESIGN: Prospective, randomised, double-blinded, placebo-controlled, parallel group, clinical trial. SUBJECTS: Forty-five adult patients presented with sudden sensorineural hearing loss, and were randomly allocated to receive either placebo (15 patients) or ozone therapy (auto-haemotherapy; 30 patients). For the latter treatment, 100 ml of the patient's blood was treated immediately with a 1:1 volume, gaseous mixture of oxygen and ozone (from an ozone generator) and re-injected into the patient by intravenous infusion. Treatments were administered twice weekly for 10 sessions. The following data were recorded: pre- and post-treatment mean hearing gains; air and bone pure tone averages; speech reception thresholds; speech discrimination scores; and subjective recovery rates. RESULTS: Significant recovery was observed in 23 patients (77 per cent) receiving ozone treatment, compared with six (40 per cent) patients receiving placebo (p < 0.05). Mean hearing gains, pure tone averages, speech reception thresholds and subjective recovery rates were significantly better in ozone-treated patients compared with placebo-treated patients (p < 0.05). CONCLUSION: Ozone therapy is a significant modality for treatment of sudden sensorineural hearing loss; no complications were observed.

Is there an association between lifetime cumulative exposure and acute pulmonary responses to ozone?

OBJECTIVE: To investigate the potential effects of lifetime cumulative ozone (O3) exposure on acute pulmonary responses to O3. METHODS: Fifteen healthy subjects from a larger cohort of young adults were exposed to 200 ppb O3 for 4 hours followed by bronchoscopy and bronchoalveolar lavage 18 hours later. Lung function, symptom questionnaires, and blood samples were obtained before and after each exposure. Subjects' lifetime cumulative O3 exposures were estimated from residential histories and air-quality monitoring data. RESULTS: Acute exposure to O3 caused decrements in forced expiratory volume in 1 second (FEV1), maximal mid-expiratory flow rate (FEF25-75), and forced expiratory flow rate at 75% of forced vital capacity (FEF75), and an increase in plasma clara cell protein (CC16) level. Changes in CC16 and lower respiratory symptoms, but not in lung function, were positively correlated with lifetime cumulative O3 exposure. CONCLUSION: Higher lifetime cumulative O3 exposure was associated with airway injury and respiratory symptom responses, but not with airway inflammatory or lung function responses, to acute O3 exposure.

Oxygenation-ozonation of blood during extracorporeal circulation: in vitro efficiency of a new gas exchange device.

We have investigated the performance of a new gas exchange device (GED), named L001, specifically devised for the ozonation of human blood during extracorporeal circulation. This procedure, defined with the acronym "EBOO," means "extracorporeal blood oxygenation-ozonation." The innovative GED is made of microporous, ozone-resistant, polipropylene hollow fibers with an external diameter of 200 microm, a thickness of 50 microm, and a membrane surface area of 0.22 m(2). The material is coated with phosphorylcholine on the external side in contact with the circulating blood, while a gas mixture, necessarily composed of medical oxygen and ozone (about 99 and 1%, respectively), flows inside the fibers in opposite direction. The new GED has been tested by using a buffered saline solution containing KI and by varying several parameters, and it has shown to be very versatile and efficient. Its main characteristics are minimal foreign surface contact, high gas transfer, and negligible priming volume. This device appears to be a practical, nontoxic, and rather inexpensive tool for performing ozonation of blood for already defined human diseases.