Effects of environmental exposure to iron powder on healthy and elastase-exposed mice

This study demonstrates that environmental exposure significantly promoted in inflammation, oxidative stress, and remodeling in healthy animals by potentially acting on NFkB. Moreover, it is interesting that these changes were similar to those observed in animals with elastase-induced emphysema that were not exposed to environmental exposure to iron powder.

Only the local pollution from Place 1 induced an inflammatory response, and this response was exacerbated when it was associated in animals that already had an inflammatory condition induced by emphysema.

A geochemical study of the region was performed during the different seasons to characterize the particulate material. Atmospheric particulate matter was sampled at the same location during the summer and winter seasons. In both summer and winter, the mean PM concentrations at Place 1 were higher than those at Place 2. During the summer period, the PM₁₀ concentrations were 164 ± 112 μg m⁻³ and 33.6 ± 12.1 μg m⁻³ at Place 1 and Place 2, respectively. During the winter period, the PM₁₀ concentrations were 51.2 ± 27.8 μg m⁻³ and 36.6 ± 13.0 μg m⁻³ at Place 1 and Place 2, respectively. The results also showed that the coarse fraction represented approximately 80% of PM_10, and for both collection sites and seasons, coarse PM concentrations were higher than fine PM concentrations.

The PM_2.5 concentrations obtained in this study were lower than the mean values obtained by Miranda et al. (2012) in Brazilian cities such as São Paulo (28.1 ± 13.6 µg m⁻³), Rio de Janeiro (17.2 ± 11.2 µg m⁻³), Belo Horizonte (14.7 ± 7.7 µg m⁻³), Curitiba (14.4 ± 9.5 µg m⁻³) and Porto Alegre (13.4 ± 9.9 µg m⁻³).

Furthermore, in both summer and winter, the mean concentrations at Place 1 were higher than those at Place 2. PM enters the human body through inhalation, and prolonged exposure to PM can worsen lung inflammation due to its direct toxic effects and production of oxidative stress².

It should also be noted that, in addition to the PM, chemical composition is a key determinant of the inflammatory response⁵. Considering the particulate matter elemental composition, we have selected chlorine (Cl), iron (Fe), sodium (Na) and sulfur (S) for discussion since they showed the highest concentrations. In the winter, chlorine concentrations were higher at Place 2 than at Place 1 in both coarse and fine PM fractions.

Increased exposure 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, have been linked to an increase in mortality in COPD patients³⁰.

There was no difference in lung mechanics among the groups in either summer or winter mice. Nonetheless, there were low to moderate correlations between lung tissue elastance and resistance and indicators of inflammation, oxidative stress, and remodeling. Hantos et al. 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, suggesting that lung tissue destruction is not always linked to lung system dysfunction³¹.

Mice exposed to fine particulate matter inhalation for four hours showed a slight but not significant increase in respiratory elastance and resistance³². 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 exposure³³.

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.

The presence of chlorine in the atmospheric PM in Vitoria may have influenced the inflammatory response and remodeling. Exposure to elevated levels of chlorine in the environment is associated with an increased occurrence of lung inflammation due to chlorine’s ability to react with respiratory mucous membranes and trigger inflammatory responses in the respiratory system. De Genaro et al. discovered that both acute and chronic exposure to chlorine gas reduces lung function and increases oxidative stress and mucus secretion in healthy mice²⁶. 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^34,35,36.

When a proinflammatory response is activated, reactive oxygen species (ROS) and proinflammatory cytokines such as TNF-α, IL-1β and interferon-gamma (INF-γ) are released, which activate iNOS^37,38. At the site of inflammation, this enzyme produces nitric oxide (NO), which increases oxidative stress³⁹. Proinflammatory 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 production⁴⁰.

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) signaling. Similar responses were observed in healthy animals exposed to low concentrations of chlorine⁴¹.

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 PM⁴², 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 iron⁴³.

Additionally, the presence of chlorine in the atmosphere can exacerbate this cytogenotoxic action. Chlorine, when combined with certain metallic components in PM, can lead to the formation of highly reactive chlorine radicals and further enhance oxidative stress in cells. This combined action of metallic components and chlorine can contribute to the development of respiratory inflammation and other health effects in individuals exposed to polluted air⁴⁴.

Through the Haber–Weiss and Fenton reactions, iron particles stimulate the production of hydroxyl radicals, which causes oxidative stress in cells^43,45. 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 (PAHs) and nitro-PAHs. 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 in DNA⁴⁵.

When compared to the animals that remained in a vivarium, the animals exposed in Vitória showed a significant increase in the in the number of positive cells of all remodeling markers. Long-term chronic exposure to PM resulted in impaired lung function, emphysematous lesions, airway inflammation, and airway wall remodeling. Exposure to PM significantly increases the expression of MMP9, MMP12, fibronectin, collagen, and TGF-β1 proteins, regardless of concentration⁴¹. Evidence has revealed that several PM components can cause cellular harm, which in turn can activate pathways for extracellular matrix remodeling^46,47. Airway remodeling refers to structural and extracellular matrix (ECM) changes in large and small airways⁴⁸. Previous studies have reported that the ECM of airway cells is altered in asthmatic patients, with a decrease in type IV collagen and elastin levels and an increase in type I collagen, fibronectin, laminin, periostin, versican, and decorin levels and lumican deposition^48,49.

Proinflammatory factors such as cytokines and proteases are secreted, which further triggers 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 process⁵⁰.

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 type for the lungs⁴¹. These particles can remain 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 attacks¹⁰.

Chan et al. discovered an increase in lymphocytes and macrophages, which was also observed in our mouse group exposed 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 levels was linked to the activation of the NLRP3 inflammasome⁴². In another study, daily exposure to 50 µg of PM_2.5 for three weeks increased both IL-1β and TGF-β1 levels in bronchoalveolar lavage fluid⁵¹.

According to Chu et al., PM_2.5 inhalation can exacerbate macrophage-induced damage 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. Researchers have concluded that PM_2.5 can upregulate the expression of genes encoding TNF-α, IL-6, and IL-1β⁵².

Increased exposure to PM_2.5 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 tissue³³. MUC5AC levels increased in mice at both exposure locations, both in the SAL-L1 and SAL-L2 control groups and in the ELA-L1 and ELA-L2 elastase groups, with MUC5AC levels being higher in the ELA-L1 and ELA-L2 groups than in the other groups.

Wang et al. discovered that PM_2.5 has a substantial impact on exacerbating COPD symptoms. According to the findings, PM_2.5 causes increased oxidative stress, airway inflammation, and goblet cell hyperplasia, which leads to imbalanced protease/antiprotease levels and airway remodeling. PM_2.5 deposited in the pulmonary bronchioles and alveoli causes oxidative stress, which initiates a chain reaction of harmful processes such as protease activation and increased bronchial inflammation, resulting in increased mucus hypersecretion, small airway fibrosis, and collagen accumulation⁴².

As a result, there is persistent inflammation and the development of pulmonary emphysema⁴². Feng et al. discovered that mice exposed to high levels of PM_2.5 for four weeks had poor lung function, mucus hypersecretion, and high levels of proinflammatory cytokines and oxidative stress indicators. According to the authors, four weeks may be sufficient time to achieve the histological changes caused by PM inhalation³³.

We observed an increase in iron deposition in alveolar macrophages in healthy, elastase-exposed mice. Although we did not observe a correlation with this iron deposition and functional changes. We noted a moderate correlation between inflammation, remodeling, oxidative stress and NFkB with the number of iron-positive macrophages. Seaton et al. demonstrated that dust in the London Underground had cytotoxic and inflammatory potential at high doses, which was consistent with the iron oxide found in the dust⁵³. 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 cells⁵⁴.

Some free radicals generated from oxidative stress have been shown to activate specific protein transcription factors, including NFkB, which upregulates the expression of genes for cytokines, chemokines, and other inflammatory mediators, as well as apoptosis- and necrosis- related genes in macrophages and respiratory epithelial cells, impairing immune defense processes and increasing airway reactivity^55,56. In this study, NFkB levels were higher in the SAL-L1, SAL-L2, ELA-L1, and ELA-L2 groups than in the SAL and ELA groups and in the ELA-L1 and ELA-L2 groups than in the SAL-L1 and SAL-L2 groups.

Increased NFkB activation can result in excessive T-cell activation, which is linked to autoimmune and inflammatory responses⁵⁷. Activated CD4 + cells differentiate into various types of effector T cells (Th1, Th2, Th17, and follicular T cells) that produce cytokines and influence immune responses⁵⁸. Inflammatory Th1 and Th17 cells are closely linked to IFN-γ secretion, which serves as a cellular immune defense and plays a role in inflammatory processes⁵⁹. IL-17, a well-known inflammatory cytokine that attracts monocytes and neutrophils to the site of inflammation, is also released by Th17 cells⁶⁰.

The use of an experimental model of exposure in different locations may have been a limitation for the study, as the animals were transported between the states of SP and ES, and even though it was a short journey, the movement can induce stress and increase the cortisol levels of these animals, and these levels were not measured in the present study.

Nevertheless, the findings of this study emphasize the importance of investigating the effects of particulate matter exposure on lung tissue. This study aimed to better understand these processes by examining inflammation, changes in the extracellular matrix, oxidative stress activation, and the signaling pathways responsible for these lung injury mechanisms.

Cellular expression of iNOS and Gp91ohox were deemed as appropriate marker in this study’s evaluation of oxidative stress because elevated levels of this marker indicated oxidative stress in healthy animals and exacerbation in animals with emphysema. Chronic exposure to high concentrations of PM₁₀ and PM_2.5 in the atmosphere, containing substantial amounts of iron and chlorine, is associated with increased pulmonary inflammation due to the absorption of these elements into respiratory tissues. The presence of iron and chlorine in PM particles triggers the formation of reactive oxygen species (ROS) and chlorinated compounds, leading to oxidative stress in the lungs. Oxidative stress, in turn, induces chronic inflammatory responses in pulmonary tissues. Furthermore, prolonged exposure to this combination of atmospheric pollutants can promote pulmonary remodeling, including fibrosis and structural alterations, resulting in a significant deterioration of lung function. This hypothesis suggests that exposure to PM₁₀, PM_2.5, iron and chlorine can initiate a cascade of events that leads to lung damage, including chronic inflammation, oxidative stress, and pulmonary remodeling. This association is relevant for understanding the threat to respiratory health in areas with high air pollution, such as in Vitória, emphasizing the importance of air quality regulation and control, as well as the pursuit of clean energy sources to mitigate these adverse effects.