Single Ozone Exposure Research Articles

Enhancing the Antimicrobial Effect of Ozone with Mentha piperita Essential Oil

This study aimed to obtain and analyse Mentha piperita essential oil (MpEO) for the prospect of being used as an enhancement agent for the antimicrobial potential of ozone against gram-positive and gram-negative bacteria and fungi. The research was done for different exposure times, and it gained time-dose relationships and time-effect correlations. Mentha piperita (Mp) essential oil (MpEO) was obtained via hydrodistillation and further analysed by using GC-MS. The broth microdilution assay was used to determine the strain inhibition/strain mass growth by using spectrophotometric optical density reading (OD). The bacterial/mycelium growth rates (BGR/MGR) and the bacterial/mycelium inhibition rates (BIR/MIR) after ozone treatment in the presence and absence of MpEO on the ATTC strains were calculated; the minimum inhibition concentration (MIC) and statistical interpretations of the time-dose relationship and specific t-test correlations were determined. The effect of ozone on the following tested strains at maximum efficiency was observed after 55 s of single ozone exposure, in order of effect strength: S. aureus > P. aeruginosa > E. coli > C. albicans > S. mutans. For ozone with the addition of 2% MpEO (MIC), maximum efficacy was recorded at 5 s for these strains, in order of effect strength: C. albicans > E. coli > P. aeruginosa > S. aureus > S. mutans. The results suggest a new development and affinity regarding the cell membrane of the different microorganisms tested. In conclusion, the use of ozone, combined with MpEO, is sustained as an alternative therapy in plaque biofilm and suggested as helpful in controlling oral disease-causing microorganisms in medicine.

Acute Respiratory Barrier Disruption by Ozone Exposure in Mice.

Ozone exposure causes irritation, airway hyperreactivity (AHR), inflammation of the airways, and destruction of alveoli (emphysema), the gas exchange area of the lung in human and mice. This review focuses on the acute disruption of the respiratory epithelial barrier in mice. A single high dose ozone exposure (1 ppm for 1 h) causes first a break of the bronchiolar epithelium within 2 h with leak of serum proteins in the broncho-alveolar space, disruption of epithelial tight junctions and cell death, which is followed at 6 h by ROS activation, AHR, myeloid cell recruitment, and remodeling. High ROS levels activate a novel PGAM5 phosphatase dependent cell-death pathway, called oxeiptosis. Bronchiolar cell wall damage and inflammation upon a single ozone exposure are reversible. However, chronic ozone exposure leads to progressive and irreversible loss of alveolar epithelial cells and alveoli with reduced gas exchange space known as emphysema. It is further associated with chronic inflammation and fibrosis of the lung, resembling other environmental pollutants and cigarette smoke in pathogenesis of asthma, and chronic obstructive pulmonary disease (COPD). Here, we review recent data on the mechanisms of ozone induced injury on the different cell types and pathways with a focus on the role of the IL-1 family cytokines and the related IL-33. The relation of chronic ozone exposure induced lung disease with asthma and COPD and the fact that ozone exacerbates asthma and COPD is emphasized.

Mitochondrial ROS and NLRP3 inflammasome in acute ozone-induced murine model of airway inflammation and bronchial hyperresponsiveness

Oxidative stress is a key mechanism underlying ozone-induced lung injury. Mitochondria can release mitochondrial reactive oxidative species (mtROS), which may lead to the activation of NLRP3 inflammasome. The goal of this study was to examine the roles of mtROS and NLRP3 inflammasome in acute ozone-induced airway inflammation and bronchial hyperresponsiveness (BHR). C57/BL6 mice (n = 8/group) were intraperitoneally treated with vehicle (phosphate buffered saline, PBS) or mitoTEMPO (mtROS inhibitor, 20 mg/kg), or orally treated with VX-765 (caspse-1 inhibitor, 100 mg/kg) 1 h before the ozone exposure (2.5 ppm, 3 h). Compared to the PBS-treated ozone-exposed mice, mitoTEMPO reduced the level of total malondialdehyde in bronchoalveolar lavage (BAL) fluid and increased the expression of mitochondrial complexes II and IV in the lung 24 h after single ozone exposure. VX-765 inhibited ozone-induced BHR, BAL total cells including neutrophils and eosinophils, and BAL inflammatory cytokines including IL-1α, IL-1β, KC, and IL-6. Both mitoTEMPO and VX-765 reduced ozone-induced mtROS and inhibited capase-1 activity in lung tissue whilst VX-765 further inhibited DRP1 and MFF expression, increased MFN2 expression, and down-regulated caspase-1 expression in the lung tissue. These results indicate that acute ozone exposure induces mitochondrial dysfunction and NLRP3 inflammasome activation, while the latter has a critical role in the pathogenesis of ozone-induced airway inflammation and BHR.

Interleukin-1α Mediates Ozone-Induced Myeloid Differentiation Factor-88-Dependent Epithelial Tissue Injury and Inflammation

Air pollution associated with ozone exposure represents a major inducer of respiratory disease in man. In mice, a single ozone exposure causes lung injury with disruption of the respiratory barrier and inflammation. We investigated the role of interleukin-1 (IL-1)-associated cytokines upon a single ozone exposure (1 ppm for 1 h) using IL-1α-, IL-1β-, and IL-18-deficient mice or an anti-IL-1α neutralizing antibody underlying the rapid epithelial cell death. Here, we demonstrate the release of the alarmin IL-1α after ozone exposure and that the acute respiratory barrier injury and inflammation and airway hyperreactivity are IL-1α-dependent. IL-1α signaling via IL-1R1 depends on the adaptor protein myeloid differentiation factor-88 (MyD88). Importantly, epithelial cell signaling is critical, since deletion of MyD88 in lung type I alveolar epithelial cells reduced ozone-induced inflammation. In addition, intratracheal injection of recombinant rmIL-1α in MyD88acid mice led to reduction of inflammation in comparison with wild type mice treated with rmIL-1α. Therefore, a major part of inflammation is mediated by IL-1α signaling in epithelial cells. In conclusion, the alarmin IL-1α released upon ozone-induced tissue damage and inflammation is mediated by MyD88 signaling in epithelial cells. Therefore, IL-1α may represent a therapeutic target to attenuate ozone-induced lung inflammation and hyperreactivity.

Ozone exposure induces respiratory barrier biphasic injury and inflammation controlled by IL-33

IL-33 plays a critical role in regulation of tissue homeostasis, injury, and repair. Whether IL-33 regulates neutrophil recruitment and functions independently of airways hyperresponsiveness (AHR) in the setting of ozone-induced lung injury and inflammation is unclear. We sought to examine the role of the IL-33/ST2 axis in lung inflammation on acute ozone exposure in mice. ST2- and Il33-deficient, IL-33 citrine reporter, and C57BL/6 (wild-type) mice underwent a single ozone exposure (1ppm for 1hour) in all studies. Cell recruitment in lung tissue and the bronchoalveolar space, inflammatory parameters, epithelial barrier damage, and airway hyperresponsiveness (AHR) were determined. We report that a single ozone exposure causes rapid disruption of the epithelial barrier within 1hour, followed by a second phase of respiratory barrier injury with increased neutrophil recruitment, reactive oxygen species production, AHR, and IL-33 expression in epithelial and myeloid cells in wild-type mice. In the absence of IL-33 or IL-33 receptor/ST2, epithelial cell injury with protein leak and myeloid cell recruitment and inflammation are further increased, whereas the tight junction proteins E-cadherin and zonula occludens 1 and reactive oxygen species expression in neutrophils and AHR are diminished. ST2 neutralization recapitulated the enhanced ozone-induced neutrophilic inflammation. However, myeloid cell depletion using GR-1 antibody reduced ozone-induced lung inflammation, epithelial cell injury, and protein leak, whereas administration of recombinant mouse IL-33 reduced neutrophil recruitment in Il33-deficient mice. Data demonstrate that ozone causes an immediate barrier injury that precedes myeloid cell-mediated inflammatory injury under the control of the IL-33/ST2 axis. Thus IL-33/ST2 signaling is critical for maintenance of intact epithelial barrier and inflammation.

Three days after a single exposure to ozone, the mechanism of airway hyperreactivity is dependent on substance P and nerve growth factor.

Ozone causes persistent airway hyperreactivity in humans and animals. One day after ozone exposure, airway hyperreactivity is mediated by release of eosinophil major basic protein that inhibits neuronal M(2) muscarinic receptors, resulting in increased acetylcholine release and increased smooth muscle contraction in guinea pigs. Three days after ozone, IL-1β, not eosinophils, mediates ozone-induced airway hyperreactivity, but the mechanism at this time point is largely unknown. IL-1β increases NGF and the tachykinin substance P, both of which are involved in neural plasticity. These experiments were designed to test whether there is a role for NGF and tachykinins in sustained airway hyperreactivity following a single ozone exposure. Guinea pigs were exposed to filtered air or ozone (2 parts per million, 4 h). In anesthetized and vagotomized animals, ozone potentiated vagally mediated airway hyperreactivity 24 h later, an effect that was sustained over 3 days. Pretreatment with antibody to NGF completely prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone and significantly reduced the number of substance P-positive airway nerve bundles. Three days after ozone, NK(1) and NK(2) receptor antagonists also blocked this sustained hyperreactivity. Although the effect of inhibiting NK(2) receptors was independent of ozone, the NK(1) receptor antagonist selectively blocked vagal hyperreactivity 3 days after ozone. These data confirm mechanisms of ozone-induced airway hyperreactivity change over time and demonstrate 3 days after ozone that there is an NGF-mediated role for substance P, or another NK(1) receptor agonist, that enhances acetylcholine release and was not present 1 day after ozone.

Lung inflammation and thrombogenic responses in a time course study of Csb mice exposed to ozone

Ozone is a well-known oxidant air pollutant, inhalation of which can result in oxidative stress, and lead to pulmonary inflammation. The aim of this study was to evaluate the time-course events after a single ozone exposure in transcription-coupled repair defective Csb and wild type mice. Mice were exposed for 3 h to 2 ppm ozone and biological parameters related to oxidative stress and inflammation were examined in the lungs at 0, 4, 9, 24 and 48 h after exposure. In addition the procoagulant and thrombomodulin activities were explored by a combination of assays for tissue factor and thrombin generation. This study revealed a significant biological response to ozone, for both Csb and wild type mice. The onset of inflammation in Csb mice, as indicated by an increase in interleukin-6, tumor necrosis factor-alpha and total cell influx, occurred earlier compared with those seen in wild type mice. On the other hand, Csb mice showed a delayed antioxidant reaction compared with wild type mice. Both genotypes developed a procoagulant reaction characterized by a stably increased tissue factor activity and a progressive increase in thrombin generation after 2 days. These experiments have shown that ozone, a well-known toxic substance from the environment, induces not only inflammation, but also procoagulant reactions in the lungs of mice. These results have implications for understanding the systemic effects induced by oxidant air pollutants.

The changing role of eosinophils in long-term hyperreactivity following a single ozone exposure

Ozone hyperreactivity over 24 h is mediated by blockade of inhibitory M(2) muscarinic autoreceptors by eosinophil major basic protein. Because eosinophil populations in the lungs fluctuate following ozone, the contribution of eosinophils to M(2) dysfunction and airway hyperreactivity was measured over several days. After one exposure to ozone, M(2) function, vagal reactivity, smooth muscle responsiveness, and inflammation were measured in anesthetized guinea pigs. Ozone-induced hyperreactivity to vagal stimulation persisted over 3 days. Although hyperreactivity one day after ozone is mediated by eosinophils, AbVLA-4 did not inhibit either eosinophil accumulation in the lungs or around the nerves or prevent hyperreactivity at this time point. Two days after ozone, eosinophils in BAL, around airway nerves and in lungs, were decreased, and neuronal M(2) receptor function was normal, although animals were still hyperreactive to vagal stimulation. Depleting eosinophils with AbIL-5 prevented hyperreactivity, thus eosinophils contribute to vagal hyperreactivity by mechanisms separate from M(2) receptor blockade. Three days after ozone, vagal hyperreactivity persisted, eosinophils were again elevated in BAL in lungs and around nerves, and M(2) receptors were again dysfunctional. At this point, airway smooth muscle was also hyperresponsive to methacholine. Eosinophil depletion with AbIL-5, AbVLA-4, or cyclophosphamide protected M(2) function 3 days after ozone and prevented smooth muscle hyperreactivity. However, vagal hyperreactivity was significantly potentiated by eosinophil depletion. The site of hyperreactivity, muscle or nerve, changes over 3 days after a single exposure to ozone. Additionally, the role of eosinophils is complex; they mediate hyperreactivity acutely while chronically may be involved in repair.

Species differences in impairment and recovery of alveolar macrophage functions following single and repeated ozone exposures

Effects of single (0.4 ppm for 3, 6, or 12 hr) and repeated (0.4 ppm, 12 hr/day for 3 or 7 days) in vivo ozone exposures on rat and mouse alveolar macrophage functions and cell number were investigated. Single ozone exposure of rats resulted in a small (∼15%) decrease in Fc-receptor-mediated phagocytosis and phorbol ester-induced superoxide production by the alveolar macrophages and was followed by recovery above control levels within 12 hr of exposure. Repeated exposures of rats for up to 7 days did not alter alveolar macrophage functions, with the exception of the effects of 3 days of exposure on superoxide production (71 ± 9% as compared with the controls). In mice, significant changes in alveolar macrophage functions were not observed until 12 hr of exposure (at that timepoint phagocytosis was 74 ± 2%). Repeated ozone exposures of mice did not cause a further decrease in phagocytosis (at Day 7, 74 ± 14%). Both after 3 and 7 days of repeated ozone exposure of mice, superoxide production by the alveolar macrophages was inhibited ∼50%. In rats and mice, repeated ozone exposures led to an increase in the number of alveolar macrophages. In mice, this increase appeared at a later time point (at Day 7 vs Day 3) and was less pronounced (at Day 7, 139 ± 9% vs 179 ± 17%) as compared with rats. In summary, our data show that rat and mouse alveolar macrophages have different susceptibilities to both single and repeated in vivo ozone exposures.

Effect of repetitive ozone treatment on bean plants—stress ethylene production and leaf necrosis

Bean plants were repetitively treated with varying ozone (O 3) concentrations and exposure times on successive days. Our studies clearly demonstrate that a series of successive short episodes of ozone exposure is more damaging to the plants than continuous exposure of the same O 3 concentration for the same total exposure time. In contrast to single ozone exposure experiments repetitive ozone treatment causes stress ethylene production and the beginning of leaf necrosis at the same time. The “no effect levels” estimated for O 3 with single exposure experiments can not be applied to repetitive ozone exposure episodes because the plants are more sensitive under these conditions.

Destruction of enzymatic activities of corn and soybean leaves exposed to ozone

Experiments were conducted to determine the effects of a single ozone exposure on selected enzymatic activities and chlorophyll contents of corn and soybean seedlings. Both nitrite reductase activity and chlorophyll content of the seedlings were found to be quite sensitive to ozonation and were seen to decrease as much as 50% after exposure to 80 parts per hundred million (pphm) ozone. After exposure to lower levels of ozone, less-pronounced decreases were observed. Nitrate reductase activity was reduced only after exposure of seedling leaf tissues to high concentrations of ozone. These results are discussed in relation to the concept of a two-phase response to oxidant exposure. The first phase is at the chloroplast level and is quite sensitive to the low as well as the high concentrations of ozone; the second is at the cellular level and is relatively resistant to all but the highest ozone concentrations.