Many studies associate events of urban air pollution with significant health effects on the exposed population, including morbidity and mortality due to cardiopulmonary diseases or lung cancer (Dominici et al., 2006; Fajersztajn et al., 2013). These outcomes have been observed even at pollution levels below current national and international ambient air quality health standards (Lin et al., 1999).

Elevated levels of air pollution in São Paulo (Brazil) have been associated with increased respiratory emergency visits, hospital admissions and even death among children and elderly people (Saldiva et al., 1994, 1995; Lin et al., 1999, 2004; Farhat et al., 2005; Atkinson et al., 2014). These results are in accordance with experimental data from air pollution studies in São Paulo. Acute exposure to diesel and traffic-derived particles impairs lung impedance, pulmonary inflammation and histology in mice (Pereira et al., 1995, 2011; Laks et al., 2008; Mazzoli-Rocha et al., 2008; Zanchi et al., 2010; Riva et al., 2011; Zin et al., 2011). In addition, long-term mice exposure to traffic-derived particulate matter (PM) yielded worse pulmonary function, bronchial/alveolar lesion, lung macrophage influx, and oxidative stress (Mazzoli-Rocha et al., 2014), secretory cell hyperplasia and ultrastructural ciliary alterations of the airway epithelium (Saldiva et al., 1992a), compromised respiratory defenses (Lemos et al., 1994), as well as cardiopulmonary oxidative damage (Damiani et al., 2012).

Residual oil fly ash (ROFA) consists of PM produced by oil-burning systems and is rich in transition metals. It has been used in murine models as a surrogate material to investigate the responses to PM inhalation (Dreher et al., 1997; Kodavanti et al., 1998). ROFA administration via intratracheal/intranasal instillation and aerosol inhalation disclosed functional and structural alterations such as acute lung injury, alveolar septal thickening, increased cellularity and lung inflammation (Dreher et al., 1997; Gavett et al., 1997, 1999; Ghio et al., 2002; Hamada et al., 2002; Kodavanti et al., 2002). Additionally, ROFA exposure has been studied in association with chronic allergic pulmonary inflammation, cigarette smoke, and lung infection (Gavett et al., 1999; Antonini et al., 2002; Arantes-Costa et al., 2008; Biselli et al., 2011). We previously reported that chronic allergic mice exposed to ROFA show even higher hyperresponsiveness, bronchoconstriction and mast cell infiltration after methacholine challenge than those not exposed (Avila et al., 2011). However, so far the timeline of the lung alterations following a single exposure to ROFA particles has not been reported.

Thus, we aimed to investigate the time-dependency of lung impairment in animals that underwent a single exposure to ROFA, simulating the situation of someone visiting a polluted place for a day. For such purpose, we analyzed ROFA composition, lung mechanics, alveolar collapse, inflammatory cells recruitment, and pulmonary ultrastructure in different time points after exposure.

ROFA analysis showed the presence of metal elements, such as copper, cadmium, chromium, nickel, manganese, lead, zinc and mainly iron (Table 1), and PAHs, such as naphthalene, acenaphthylene, fluorene, acenaphthene, antracene, flouranthene, phyrene, benzo[k]fluorantene, benzo[ghi]peryle (some with carcinogenic potencial: benzo[a]antracene, benzo[a]pyrene, Dbenzo[ah]antracene and ind[123cd]pyrene) (Table 2). Organochloride elements as g-hexachlorocyclohexane (g-HCH), endosulfan, dieldrin, op'-DDE (dichlorodiphenyl dichloroethylene), op'-DDT (dichlorodiphenyltrichloroethane), pp'-DDT were also present (Table 3).

Flows and inspired volumes did not differ among groups. Figure 1 shows ΔP's, obtained in CTRL, ROFA6, ROFA24, ROFA48, ROFA72, ROFA96, and ROFA120 groups. ΔP1 augmented early (6 h) in the course of lung injury (184%) and remained elevated until 96 h (137%). ΔP2, Est and ΔE increased in ROFA24 (70, 88, and 68% respectively) and remained elevated until 96 h (68, 50, and 64% respectively). On the fifth day (ROFA120) all parameters returned to CTRL values (Figures 1, 2).

The fraction area of alveolar collapse and PMN cell influx into the lung parenchyma were higher in ROFA than in CTRL at 24, 48, 72, and 96 h. Similarly, normal alveolar spaces and MN cells were significantly lower in the latter groups than in CTRL. ROFA120 showed values similar to CTRL for histological parameters (Figures 3, 4). ROFA particle was observed in alveolar space of ROFA6, 24, 48, 72, and 96 groups and not in ROFA120 (Figure 3, insert B).

Electron microscopy of lung parenchyma in CTRL mice showed preserved types I and II pneumocytes, endothelial cells, alveolar interstitial wall, and components of the extracellular matrix. At 24 h, ROFA group showed endothelial damage as represented by swelling, vacuolization and neutrophils adhered to the pulmonary capillary wall (Figure 5).

The amount of neutrophils in the blood was higher in ROFA24 and ROFA48 than in the CTRL mice. Thereafter they did not differ from CTRL animals. No statistically significant difference was detected for neutrophils in the BALF (Figure 6).

The survival rate was 100% in all groups throughout the experiments.

The time course of lung functional and histological impairment induced by ROFA dust has not been reported so far. Intranasal instillation of a low dose of ROFA (0.2 μg in 10 μL) induced a significant increase in resistive pressure, followed by an increment in viscoelastic/inhomogeneous pressures and elastances, accompanied by increased alveolar collapse, influx of PMN cells, ultrastructural alterations in lung parenchyma and increased number of neutrophils in the blood. These outcomes returned to control values at 120 h after exposure.

PM is a heterogeneous mixture of gas, liquid, and solid particles of different origins and sizes in suspension in the air, displaying close physical and chemical interactions. PM is classified, according to its aerodynamic diameter, as coarse (2.5–10 μm; PM10), fine (0.1–2.5 μm; PM2.5), and ultrafine (≤0.1 μm) (Donaldson et al., 2001). The different profiles of size and composition may influence particle toxicity and, consequently, the magnitude of adverse health effects (Saldiva et al., 2002). In human beings, toxicity becomes very important when aerodynamic diameter of the particles is 10 μm, which enables them to reach the pulmonary alveoli. In rats and mice, this value approximates 2 μm for intratracheally instilled silica (Wiessner et al., 1989; Takayoshi et al., 2007). The ROFA used in the present study was mainly composed of particles bigger than 10 μm (Avila et al., 2011), which would be less harmful than the smaller ones (Donaldson et al., 2001). However, around 7.6% of ROFA particles presented an average diameter less than 10 μm, and about 2.1% were smaller than 2.5 μm. Another concomitant study of our group (Avila et al., 2011), using the same dose and PM, reported lung impairment at 24 h after exposure. It should be stressed that the fine and ultrafine particles are known as “breathable” and are able to penetrate the airways, reaching the alveoli (Dusseldorp et al., 1995; Peters et al., 1997; Brown et al., 2002; Tao et al., 2003). Indeed, in ROFA6, 24, 48, 72, and 96 groups the administered pollutant was detected in the alveolar space (Figure 3).

A recent study analyzed the composition, sources and toxicity of PM2.5 collected in different cities in the United States and observed an association between its toxicity and the number of vehicles and industries (Seagrave et al., 2006). São Paulo is the most industrialized center of Latin America and has about 20,000,000 inhabitants. There are about 7,000,000 vehicles in the area using three types of fuel: gasoline, diesel and alcohol (CETESB, 2013). Because of its geographical characteristics, São Paulo presents thermal inversions, resulting in significant increases in air pollution. Thus, São Paulo represents an excellent place to assess the effects of air pollution on health.

In order to avoid the consequences of particle overload, we administered a low dose of PM to the mice, reflecting more precisely the adverse pulmonary consequences of ambient particle concentrations. The mean daily concentrations of PM2.5 and PM10 in São Paulo, where ROFA was collected, amount to 60 and 120 μg/m³, respectively (CETESB, 2013). Considering that a mouse inspires 0.03 m³ of air in 24 h, it represents 6.7 μg/m³ of ROFA dust in the present work. Particles were administered by intranasal instillation to the anesthetized animal, which is a useful and well-accepted model of exposure to PM (Southam et al., 2002). In experimental models similar to ours, which used ROFA instilled intranasally in mice, doses amounting to 25 times (Biselli et al., 2011; Magnani et al., 2013; Marchini et al., 2014) and up to 60 times greater (Arantes-Costa et al., 2008) than ours were used.

ROFA is a suspension of the material produced after oil burning, which was used in some experimental models of exposure to air pollution (Ghio et al., 2002; Arantes-Costa et al., 2008; Avila et al., 2011; Marchini et al., 2014). Although ROFA exposure does not exactly mimic the overall environmental pollution, this PM contains high concentrations of many components of air pollution. Previous studies report that PMs from different sources, including ROFA (Kodavanti et al., 1998), are able to induce inflammatory processes (Sørensen et al., 2003; Park et al., 2006). Animal studies demonstrate that the bioavailability of soluble transition metals is responsible for the pulmonary injury and inflammation observed after ROFA exposure (Dreher et al., 1997; Kodavanti et al., 1998). The ROFA used in the present work contains predominantly iron and nickel (Table 1), in line with other studies. Metals, including iron, vanadium, and nickel, are present in high concentrations as water-soluble salts in fly ash (Schroeder et al., 1987) and largely reproduce the lung injury induced by ROFA. Without those metals pulmonary toxicity decreases (Dreher et al., 1997). ROFA containing iron, aluminum, vanadium and nickel induced more pronounced cellular oxidative imbalance and lung injury (Lewis et al., 2003). Iron is deeply linked to the generation of reactive oxygen species (ROS) (Park et al., 2006), surfactant dysfunction (Chauhan and Misra, 1991), epithelial damage, increased vascular permeability and inflammatory response followed by impaired pulmonary function (Soukup et al., 2000; Dye et al., 2001).

In the present study, PM concentration of PAH, particularly naphthalene, acenaphthylene, acenaphthene and other elements with potential carcinogenic risk (benzo[a]antracene, benzo[a]pyrene, Dbenzo[ah]antracene and ind[123cd]pyrene) were detected (Table 2). Another study of our group, analyzing particles produced by traffic, detected benzo[a]pyrene and benzo[a]anthracene in samples of total suspended PM from São Paulo, confirming the high levels of PAH in this city (Mazzoli-Rocha et al., 2008). Washing the diesel particles with hexane removed a large amount of PAHs and improved respiratory outcomes in mice (Laks et al., 2008). Finally, the presence of PAH in ROFA has been associated with the triggering of inflammation, generation of ROS, and lipid peroxidation (Sørensen et al., 2003), especially in alveolar macrophages and epithelial cells (Li et al., 2002).

ROFA was tested for the presence of some organochlorine compounds, and a few were found: op'-DDE, pp'-DDT, op'DDT, G-HCH, endosulfan and dieldrin (Table 3). These substances are pesticides and constitute a family of persistent, lipophilic compounds whose use was banned because they cause a variety of diseases in humans and wildlife (Androutsopoulos et al., 2013). The chlorinated pesticides may be absorbed into the body through the skin, respiratory and digestive tracts (Yohannes et al., 2014). Organochlorines cause neurotoxic, hormonal, immuno-modulating, and tumorigenic effects (Androutsopoulos et al., 2013). However, to our knowledge, no study evaluated the association between exposure to organochlorines and pulmonary impairment.

Although most of lung changes in mechanical properties, histology and inflammatory response occured at time point of 24 h, our control group was studied at 6 h after exposure. To support our approach, we compared our CTRL group with that previously reported and measured at 24 h after exposure to ROFA (Avila et al., 2011), Their results are very similar to ours, thus allowing the use of a 6-h CTRL group. Furthermore, we coped with Russel and Burch's (1959) principle of the 3Rs (reduction, refinement, and replacement of the animal use) to minimize the number of experimental animals.

A higher resistive pressure (that reflects Newtonian or ohmic resistance) was the first response to ROFA, as found in ROFA6 group (Figure 1). It can be possibly explained anatomically, since central airways are the first lung structure to be exposed to ROFA. In a previous study, we also found increased central airway resistance 24 h after exposure to ROFA; it should be noted that the authors did not perform any measurements before that time point (Avila et al., 2011). Viscoelastic and total pressures, static elastance and elastic component of viscoelasticity increased significantly in ROFA24 group and remained elevated until 4 days after exposure (ROFA96), when compared to CTRL (Figures 1, 2). We also detected higher Est and mechanical parameters related to the lung periphery 24 h after exposure to ROFA (Avila et al., 2011). These results could be explained by the concomitant increase in alveolar collapse and lung PMN content (Figures 3, 4). At the same time neutrophils adhered to the swollen pulmonary capillary wall (Figure 5), indicating activation of the endothelium and of leukocyte integrins (Langer and Chavakis, 2009), as a result of a local proinflammatory stimulus presumably triggered by ROFA. These neutrophils would migrate through the endothelium and reach the pulmonary interstitial space (Figure 4). ROFA24 also presented a higher count of PMN in the blood (Figure 6), suggesting a systemic inflammatory status. All these findings were also present in ROFA48 mice. In line with our results in ROFA24 mice, some authors observed impaired lung mechanics, alveolar collapse, influx of inflammatory cells to the lung (Avila et al., 2011), inflammatory process in the perivascular area, and inflammatory infiltration in the interstitial space (Medeiros et al., 2004). Interestingly, a recent study demonstrated increased TNF-α and IL-6 plasma levels and PMN leukocytes activation at 1, 3, and 5 h after ROFA exposure (Marchini et al., 2014), but they used doses 25 times larger than ours. Finally, it should be mentioned that surfactant secretion by type II pneumocytes is impaired after inhalation of air pollutants (Müler et al., 1998) and exposure to fly ash modifies surfactant composition (Srivastava and Misra, 1986; Chauhan and Misra, 1991) and rheology (Anseth et al., 2005), yielding alveolar instability and collapse. At 72 h after exposure blood PMN count returned to control values, but the pulmonary parameters remained higher than CTRL, suggesting that the overall process started to recede in the organism. ROFA96 presented similar results. At 120 h all measured parameters returned to baseline values.

Neutrophils count in the BALF resulted negative (Figure 6). The method may be not sensitive enough to detect the inflammatory changes after the nasal instillation of ROFA or the cells indeed did not cross the airway epithelium. In accordance with our findings, the intranasal instillation of ROFA did not disclose inflammatory alterations in mice BALF, even taking into consideration the use of a dose 500 times larger than ours (Medeiros et al., 2004). On the other hand, the intratracheal instillation of ROFA in a dose 60 times larger than that in this study triggered inflammatory alterations in BALF (Gavett et al., 1999). The difference between these two apparently discrepant results could be the local of administration of the pollutant.

Epidemiological studies can add translational information to our findings. Dose-dependent decreased indexes of pulmonary function, including diminished forced vital capacity, forced expiratory volume in 1 s, and forced expiratory flows were described in boilermakers 24 h after exposure to ROFA (Hauser et al., 1995, 1996). In a 2-year longitudinal study a significant association between working at oil-fired industries and reduced lung function was detected (Hauser et al., 2002). Finally, ROFA-exposed individuals presented impaired pulmonary function, which was resolved 4 weeks after they were removed from their working stations in an oil-fired electricity generating plant (Lees, 1980).

Our study presents some limitations: (1) the animals were exposed intranasally rather than directly to environmental air. On one hand they received only ROFA, but on the other one the results do not represent exactly what would be found around the sampling site; (2) we did not measure levels of inflammatory cytokines that could have been modified by exposure to ROFA.

In conclusion, we demonstrated that the exposure to low doses of ROFA rapidly compromised pulmonary mechanics and histology, triggered the influx of polymorphonuclear cells into the lung, and increased the neutrophil count in the blood of mice. These pathophysiological findings resolved 5 days after exposure.

Giovanna Marcella Cavalcante Carvalho—interpretation of data for the work; drafting the work and revising it for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Lilian Katiê da Silva Nagato—interpretation of data for the work, experimental design and organization, data analyses; revised the work for important intellectual content; final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Sheila da Silva Fagundes—data acquisition; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Flávia Brandão dos Santos—data acquisition; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Andrea Surrage Calheiros—data acquisition; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Olaf Malm—chemical analyses; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Patricia Torres Bozza—data analyses; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Paulo Hilário N. Saldiva—data analyses; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Débora Souza Faffe—experimental design and organization; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Patricia Rieken Macedo Rocco—experimental design and organization; revised the work for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Walter Araujo Zin—experimental design and organization, hypotheses, interpretation of data for the work; drafting the work, revised the work for important intellectual content; final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Centers of Excellence Program (PRONEX/FAPERJ), The Brazilian Council for Scientific and Technological Development (CNPq), Carlos Chagas Filho Rio de Janeiro State Research Supporting Foundation (FAPERJ), Brazilian Ministry of Science, Technology and Innovation (MCTI), and Financing for Studies and Projects (FINEP). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.