This study demonstrates that environmental exposure in the city of Vitória, ES, promoted significant changes in inflammatory markers, oxidative stress, remodeling markers, and mechanisms involved (NFkB) of healthy animals. Such changes resulted in response patterns that are similar to those of the animals with elastase-induced emphysema, kept in a vivarium in São Paulo.
In this experimental model of emphysema, most analyses revealed that inflammatory markers, oxidative stress, remodeling markers, and mechanisms involved (NFkB) were higher in exposed groups compared to control animals kept in a vivarium in São Paulo. As a result, it is possible to conclude that local pollution promoted inflammation in healthy animals while exacerbating existing inflammation in animals with emphysema.
An increase in the number of people exposed to sulfur dioxide (SO2) and nitrogen dioxide (NO2) at World Health Organization-acceptable concentrations, as well as PM less than or equal to 10 µm in aerodynamic diameter (PM10), has been linked to an increase in mortality caused by COPD exacerbation.23
In both seasons, there was no difference in lung mechanics between the groups. Nonetheless, there were low to moderate correlations between lung tissue elastance and resistance and indicators of inflammation, oxidative stress, and markers of remodeling and involved mechanisms. Hantos et al. (2008) discovered that mice given an intratracheal injection of elastase had an increase in volume and a decrease in lung tissue elastance but no change in airway and lung tissue resistance. This leads to the conclusion that lung tissue destruction is not always linked to lung system dysfunction.24
Mice exposed to fine particulate matter (PM) inhalation for four hours showed a slight but not significant increase in respiratory elastance and resistance.25 There were no changes in lung function after two weeks of exposure to PM in a high-concentration environment; changes in lung function occurred only after four weeks of exposure.26
It should be noted that the animals in this study were exposed to ambient air in three different locations for four weeks, leading to the hypothesis that the exposure time was insufficient to cause changes in pulmonary mechanics.
Parallel to the experimental model analysis, Professor Christine Bourotte conducted a geochemical study of the region during the same summer and winter seasons to characterize the particulate material present. This revealed that the coarse fraction contained approximately 80% PM10. For both collection sites and seasons, coarse PM concentrations were higher than fine PM concentrations.
Furthermore, in both summer and winter, mean concentrations at Place 1 were higher than at Place 2 (data not shown). PM enters the human body through breathing, and prolonged exposure to it can worsen lung inflammation due to its direct toxic effects and production of oxidative stress.2
It should also be noted that, in addition to the PM area, chemical composition is a key determinant of inflammatory response.5 During Professor Christine Bourotte's analysis, the elements with the highest concentrations in the particulate matter were chlorine (Cl), iron (Fe), and sulfur (S). In the winter, chlorine concentrations were higher at Place 2 than at Place 1 in both coarse and fine materials (data not shown).
De Genaro et al. (2021) discovered that both acute and chronic exposure to chlorine gas reduces lung function and increases oxidative stress and mucus secretion in healthy mice.26 When inhaled, chlorine can become solubilized in the bronchoalveolar fluid, cross the cell membrane, react with local proteins, activate local inflammation, and cause epithelial damage due to oxidative stress.27–29
When the pro-inflammatory signal is activated, reactive oxygen species (ROS) and pro-inflammatory cytokines such as TNF-α, IL-1β and interferon-gamma (INF-γ) are released, which activate induced nitric oxide synthase (iNOS).30,31 At the site of inflammation, this enzyme produces nitric oxide (NO), which increases oxidative stress.32 Pro-inflammatory cytokines can activate the Th17 response, resulting in the production of IL-17 and the recruitment of neutrophils, as well as tissue remodeling and mucus production.33
A single exposure to low doses of chlorine potentiated the Th2 response in asthmatic mice, resulting in increased inflammation, altered lung function, and activation of iNOS and kinase 2 (ROCK-2). Similar responses were observed in healthy animals exposed to low concentrations of chlorine.34
Recent research has revealed that several PM components can cause cellular harm, which in turn can activate pathways for extracellular matrix remodeling.35,36 Airway remodeling refers to structural and extracellular matrix (ECM) changes in large and small airways.37 According to research, the extracellular matrix (ECM) of the airway is altered in asthmatic patients, with a decrease in type IV collagen and elastin and an increase in type I collagen, fibronectin, laminin, periostin, versican, decorin, and lumican deposition.37,38
Pro-inflammatory factors such as cytokines and proteases are secreted, which further fuels immune responses and contributes to ECM remodeling.39,40 Numerous immune cells, including but not limited to neutrophils, eosinophils, monocytes, macrophages, and mast cells, play a role in this.39
Even though PM10 is present in higher concentrations in the air of Vitória-ES, particles with diameters smaller than 2.5 µm can penetrate the bronchioles and alveoli, making it the most dangerous particle for the lungs.34 Particles with this diameter distribution linger in the atmosphere for a longer period, increasing the likelihood of inhalation and the rate at which the composition of the air changes. The health consequences range from an increased risk of cardiovascular disease, chronic lung inflammation, and decreased lung function to an increase in asthma attacks.10
Chan et al. (2019) discovered an increase in lymphocytes and macrophages, which was also observed in exposure to high doses of PM. Nonetheless, exposure to 5 µg of PM10 did not result in the activation of eosinophil or neutrophil-driven inflammation. As expected, the increase in IL-1β was linked to the activation of NLRP3 inflammasome.42 In another study, daily exposure to 50 µg of PM2.5 for three weeks increased both IL-1β and TGF-β1 levels in bronchoalveolar lavage fluid.40
According to Chu et al. (2016), PM2.5 inhalation can exacerbate damage to macrophages in the air sacs of mice with COPD. They discovered that IL-6, IL-8, and TNF-α levels increased in bronchoalveolar lavage fluid, exacerbating airway inflammation. The researchers concluded that PM2.5 can stimulate the expression of genes encoding TNF-α, IL-6, and IL-1β.41
When compared to the animals that remained in a vivarium, the animals exposed in Vitória showed a significant increase in all remodeling markers. Long-term chronic exposure to PM2.5 resulted in impaired lung function, emphysematous lesions, airway inflammation, and airway wall remodeling. Exposure to PM2.5 significantly increases the expression of MMP9, MMP12, fibronectin, collagen, and TGF-β1 proteins, regardless of concentration.34
Increased exposure to PM2.5 can cause goblet cell hyperplasia and excessive mucus secretion in mice with COPD by increasing the expression levels of MUC5AC, MUC5B, collagen I, and collagen III in lung tissue.26 MUC5AC increased in both exposure locations, both in SAL-L1 and SAL-L2 control groups, and in ELA-L1 and ELA-L2 elastase groups, with MUC5AC being higher in the ELA-L1 and ELA-L2 groups when compared to the other groups.
Wang et al. (2020) discovered that PM2.5 has a significant impact on exacerbating COPD symptoms. According to the findings, PM2.5 causes increased oxidative stress, airway inflammation, and goblet cell hyperplasia, which leads to imbalanced protease/antiprotease levels and airway remodeling. PM2.5 deposited in the pulmonary bronchioles and alveoli causes oxidative stress, which sets off a chain reaction of harmful processes such as protease activation and increased bronchial inflammation, resulting in increased mucus hypersecretion, small airway fibrosis, and collagen buildup.42
As a result, there is persistent inflammation and the development of pulmonary emphysema.42 Feng et al. (2019) discovered that mice exposed to high levels of PM2.5 for four weeks had worsening lung function, mucus hypersecretion, and levels of pro-inflammatory cytokines and that oxidative stress indicators increased. According to the authors, four weeks may be sufficient time to achieve the histological changes caused by PM inhalation.26
When compared to the animals kept in a vivarium, those exposed in Vitória showed an increase in iNOS. The cytogenotoxic action of PM may be directly linked to oxidative stress. Several studies have been conducted to determine the cytogenotoxic action of PM,46,47 which has been primarily attributed to metallic components bound or adsorbed on particles, particularly transition metals capable of inducing the formation of reactive oxygen species (ROS), such as iron.
Through the Haber-Weiss and Fenton reactions, iron particles stimulate the production of hydroxyl radicals, which causes oxidative stress in DNA, proteins, and lipids.43,44 According to research, reactive oxygen species (ROS) can be produced on the surface of particles as a result of the absorption of polycyclic aromatic hydrocarbons (PAH) and nitro-PAH. The Fenton reaction, which is catalyzed by transition metals such as iron, copper, chromium, and vanadium, produces the highly reactive hydroxyl radical by combining Fe2+, H2O2, and H+, which can cause oxidative damage to DNA.44
Seaton et al. (2005) demonstrated that dust on the London Underground had cytotoxic and inflammatory potential at high doses, which was consistent with the dust's iron oxide composition.45 The presence of soluble metals, such as iron, nickel, vanadium, cobalt, copper, and chromium, in inhaled particles may cause an increase in cellular oxidative stress in airway epithelial cells.46
Some free radicals generated from oxidative stress have been shown to activate specific protein transcription factors, including nuclear factor kappa B (NFkB), which increases the expression of genes for cytokines, chemokines, and other inflammatory mediators, as well as apoptosis and necrosis inducers of macrophages and respiratory epithelial cells, impairing organic defense and increasing airway reactivity.47,48 In this study, NFkB levels were higher in SAL-L1, SAL-L2, ELA-L1, and ELA-L2 groups than in SAL and ELA groups, and in ELA-L1 and ELA-L2 groups than in SAL-L1 and SAL-L2 groups .
Unbalanced NFkB activation can result in excessive T cell activation, which is linked to autoimmune and inflammatory responses.49 Activated CD4 + cells differentiate into various types of effector T cells (Th1, Th2, Th17, and follicular T cells) that produce cytokines and influence immune responses.50 Inflammatory Th1 and Th17 cells are closely linked to IFN-γ secretion, which serves as cellular immunity defense and plays a role in inflammatory processes.51 IL-17, a well-known inflammatory cytokine that attracts monocytes and neutrophils to the site of inflammation, is also released by Th17 cells.52
The use of an experimental animal model was a limitation of this study because it was not possible to directly extrapolate its findings to those expected in humans. Furthermore, the animals were stressed as a result of the environmental exposure being performed in another city, forcing the study's team to make two plane trips.
Despite this, the findings of this study emphasize the importance of investigating the effects of particulate matter exposure on lung tissue. This study aims to better understand these processes by examining inflammation, changes in the extracellular matrix, oxidative stress activation, and the signaling pathway responsible for these lung injury mechanisms.
Cellular expression of iNOS was deemed an appropriate marker in this study's evaluation of oxidative stress because elevated levels of these markers indicate inflammation development in healthy animals and exacerbation in animals with emphysema.
The goal of future research is to broaden the examination of oxidative stress by incorporating additional markers and delving deeper into anti-inflammatory cytokines. Furthermore, the presence of Th1, Th2, and Th17 cytokines was investigated, as well as a thorough examination of the remodeling process, which included an examination of collagen fibers, TIMP-1, MMP-9, MMP-12, TGF-β, and the NFkB signaling pathway.