Milan PM1 Induces Adverse Effects on Mice Lungs and Cardiovascular System

Recent studies have suggested a link between inhaled particulate matter (PM) exposure and increased mortality and morbidity associated with cardiorespiratory diseases. Since the response to PM1 has not yet been deeply investigated, its impact on mice lungs and cardiovascular system is here examined. A repeated exposure to Milan PM1 was performed on BALB/c mice. The bronchoalveolar lavage fluid (BALf) and the lung parenchyma were screened for markers of inflammation (cell counts, tumor necrosis factor-α (TNF-α); macrophage inflammatory protein-2 (MIP-2); heme oxygenase-1 (HO-1); nuclear factor kappa-light-chain-enhancer of activated B cells p50 subunit (NFκB-p50); inducible nitric oxide synthetase (iNOS); endothelial-selectin (E-selectin)), cytotoxicity (lactate dehydrogenase (LDH); alkaline phosphatase (ALP); heat shock protein 70 (Hsp70); caspase-8-p18), and a putative pro-carcinogenic marker (cytochrome 1B1 (Cyp1B1)). Heart tissue was tested for HO-1, caspase-8-p18, NFκB-p50, iNOS, E-selectin, and myeloperoxidase (MPO); plasma was screened for markers of platelet activation and clot formation (soluble platelet-selectin (sP-selectin); fibrinogen; plasminogen activator inhibitor 1 (PAI-1)). PM1 triggers inflammation and cytotoxicity in lungs. A similar cytotoxic effect was observed on heart tissues, while plasma analyses suggest blood-endothelium interface activation. These data highlight the importance of lung inflammation in mediating adverse cardiovascular events following increase in ambient PM1 levels, providing evidences of a positive correlation between PM1 exposure and cardiovascular morbidity.


Introduction
Epidemiology studies have shown that increased levels of particulate matter (PM) in ambient air are associated with aggravation of respiratory diseases and cardiovascular function impairment. ese adverse events have been correlated with exposure to �ne PM (particles with aerodynamic diameter ≤2.5 m) [1], even if the pathophysiological mechanisms remain still unclear.
Lung PM penetration and clearance are size dependent: larger particles (greater than 10 m), deposited in the upper airways, are removed by the mucociliary clearance mechanism, while smaller particles (below 10 m) reach deeper the lungs and are only partially removed by alveolar macrophages (AMs) [2].
Following PM deposition in the lungs, AMs rapidly phagocytose particles and migrate towards the bronchoalveolar junction [3]. A large number of ultra�ne particles (≤0.1 m in aerodynamic diameter), however, poses a substantial burden for the macrophage phagocytic system and results in increased number of particles coming in contact with the respiratory epithelium. Damage to the capillary endothelium and type I alveolar cells has been observed as one of the earliest events in lung toxicity mediated by particles, leading to neutrophils recruitment and triggering the onset of an acute in�ammatory status [2,4]. Moreover, PM contains transition metals able to generate reactive oxygen molecules which in turn exert a cytotoxic effect on lung cells [2].
Translocation of inhaled nanoparticles across the alveolar-blood barrier has been demonstrated in animal studies for a range of nanoparticles delivered by inhalation or instillation [5][6][7]. Convincing demonstration of translocation has been difficult to achieve in humans [8,9]; however, given the deep penetration of nanoparticles into the alveoli and the close apposition of the alveolar wall and capillary network, such particle translocation seems plausible either as a naked particle or aer ingestion by AMs.
Many authors proposed the hypothesis that �ne particles inhalation provokes a low grade in�ammatory response in the lung, causing an exacerbation of preexisting lung diseases. We previously reported [10] that a single intratracheal instillation of �ne particles in mice stimulates mild lung in�ammation. e present study extends these �ndings by showing that the repeated deposition of particulate matter in the lungs triggers the onset of systemic adverse events.
Milan's particles concentration and its chemical composition have been widely examined [11,12]. Despite the number and quality of chemical data, the biological effects produced on in vivo and in vitro systems have been only recently and partly investigated [4,10,13,14]. In this paper we present and discuss pulmonary and cardiovascular adverse events induced in mice by intratracheal instillation of Milan PM1. is fraction represents PM with aerodynamic diameter ≤1 m [15], constituted by almost 40% of particles ≤400 nm in diameter. Lonati and Giugliano [16] monitored particles size distribution in four different sites in Milan and concluded that, at open air sites, 99.5% of the total number of particles are characterised by a diameter smaller than 1 m.

Animals.
Male BALB/c mice (7-8 weeks old) were purchased from Harlan Laboratories (Italy); food and water were administered ad libitum. Mice were housed in plastic cages under controlled environmental conditions (temperature 19-21 ∘ C, humidity 40-70%, lights on 7 am-7 pm). e established rules of animal care approved by Italian Ministry of Health (DL 116/92) were followed. Intratracheal instillations have been performed in mice under controlled general anaesthesia to avoid pain and discomfort. During the whole experiments we found no changes in mice weights or behaviour.

PM Sources and Chemical
Characterization. Atmospheric PM1 was collected during 2007-2008 in a Milan urban area as described in previous papers [13]. e sampling site was located at Torre Sarca, an urban site with high vehicle traffic. Samplers were located in a fenced area at about 2.5 m from the ground, 10 m from the road, and 30 m from the nearest traffic light. PM1 was sampled and chemical analyses were performed as described in Perrone et al. [17]; Milan PM1 chemical composition (inorganic ions, elements, and PAHs) is summarized in Table 1.
Particles' suspensions were prepared as follow: just before the intratracheal instillation, PM1 aliquots were properly diluted in sterile pyrogen-free saline, sonicated and vortexed, and then immediately instilled in mice.

Dose.
Our study was designed to measure the systemic response to repeated PM1 exposure and test in an animal model the hypothesis that sustained PM1 exposure could exert cardiovascular dysfunctions. Similar investigations have been previously based on very high PM exposure rate in single or repeated intratracheal instillation [20][21][22][23].
Lung in�ammation play a key role in enhancing the extrapulmonary translocation of particles [21]. So, we tested the threshold valid to lengthen the PM1 proin�ammatory effects within lungs of our BALB/c mice, and it resulted higher than the estimated daily dose possibly deposited at the hot spot of lungs in worst pollution conditions. Ideally, in vivo studies should be performed with realistic dose levels, but, as already indicated for in vitro systems [24], short-term in vivo applications have some limitations, �rst of all the necessity to obtain measurable responses within few days.
We started from the dose used by Happo et al. [22], who instilled in mice a cumulative dose of 0.82 mg/animal of �ne PM in a week, and we reduced the cumulative dose to 0.3 mg/animal of PM1 within the same time frame, in order to avoid particles lungs overload. e PM dose here used is not directly correlated to human urban PM exposures, but it has been determined as the lowest which induces a mild but still sustained lung in�ammatory response in PM1 exposed mice.

Intratracheal PM1 Instillation and Broncho Alveolar
Lavage. Animal testing was carried out by instilling 4 mice for each experimental group and the experiment was replicated twice, for a total of 8 sham and 8 PM1-treated mice.
Male BALB/c mice were brie�y exposed to a mixture of 2.5% iso�urane (�urane) anesthetic gas and kept under anaesthesia during the whole instillation procedure. Once a deep stage of anaesthesia was reached, mice were intratracheally instilled by means of MicroSprayer Aerosolizer system (MicroSprayer Aerosolizer-Model IA-1C and FMJ-250 High Pressure Syringe, Penn Century, USA) with 100 g of PM1 in 100 L of isotonic saline solution, or 100 L of isotonic saline solution (sham) as previously reported [4,10,14]. Each mouse was placed in a supine position, the mouth was opened and the tongue was gently moved aside using a pince to better incannulate the trachea. e particles were suspended in the appropriate solution just before the intratracheal instillation. PM1-treated and sham mice were allowed to recover under visual control before placing them back in plastic cages, under controlled environmental conditions. e intratracheal instillation was performed on days 0, 3, and 6, for a total of three instillations. 24 h aer the last instillation, mice from each experimental group were euthanized T 1:  [18]. Concerning sources, traffic and heating during cold season constitute the 49-53% of the primary combustion sources of �ne PM; during warm season they constitute about the 25%, while secondary sources are predominant (50-66%) [19]. Elemental carbon (primarily from traffic) contributes for about 10-15% to the �ne fraction; organic matter, calculated applying a speci�c organic matter-to-organic carbon conversion factor to each source, contributes for 31-38% to the �ne fraction [19]. with an anesthetic mixture overdose (Tiletamine/Zolazepam-�ylazine and iso�urane). e effects were assessed 24 h aer the last treatment since the greatest in�ammatory response occurs by this time [22]. e broncho alveolar lavage (BAL) procedure, pellets, and supernatant recovery have been performed as described in Mantecca et al. [4,14].

LDH and ALP.
LDH and ALP assays were performed on cell-free BALf supernatants. e commercially available kits for alkaline phosphatase (DALP-250 QuantiChrom Alkaline Phosphatase Assay Kit, Gentaur Molecular) and lactate dehydrogenase (DLDH-100 QuantiChrom Lactate Dehydrogenase Kit, Gentaur Molecular) were employed according to the manufacturers' instructions.

2.5.3.
Cytokines. e analyses of proin�ammatory cytokines released in the BALf was performed by DuoSet ELISA kits for tumour necrosis factor-and macrophage in�ammatory protein-2 (TNF-and MIP-2; R&D Systems) according to the manufacturer's protocols.
2.6. Lung and Heart Biochemical Analyses. e lungs of sham and PM1-treated mice, at the end of BAL procedure, were quickly excised from the chest and washed in ice cold isotonic saline solution. e le lobes were dissected and submitted to histology, the right lobes were preserved for the biochemical analyses. For protein assays, lungs were minced at 4 ∘ C, suspended in NaCl 0.9%, brie�y homogenized for 30 seconds at 11000 rpm with Ultra-Turrax T25 basic (IKA WERKE) and sonicated for other 30 seconds. en samples were submitted to trichloroacetic acid (TCA) precipitation according to the procedure described in Farina et al. [10]. e pellets were suspended in water and protein quantity determined by BCA method (Sigma Aldrich, USA).
ereaer, lung homogenates of sham and PM1-treated mice were loaded on SDS-PAGE and submitted to electrophoresis, followed by Western blot. e membranes were stained with Ponceau and the protein loading was assessed by densitometry (BIORAD Densitometry 710, program Quantity one) as described [25]. Aer blocking, blots were incubated for 2 h with the primary antibody diluted in PBS-Tween20/milk. Lung parenchyma was tested for Cyp1B1, HO-1, Caspase8-p18, NF B-p50, Hsp70, E- Proteins were detected by ECL using the SuperSignal detection kit (Pierce, Rockford, IL). Immunoblot bands were analyzed and the optical density (OD) quanti�ed by KODAK (Kodak Image Station 2000R); all the data have been normalized to -actin (anti--actin 1 : 1500 by Sigma) and each protein in PM1-treated group was normalized to the corresponding sham group.
Heart tissue from sham and PM1-treated mice was submitted to all the procedures above described for lungs, and homogenates tested for HO-1, Caspase8-p18, NF B-p50, Eselectin, iNOS, and MPO (anti-HO-

Lung Histopathological Analyses.
Once excised, the le lungs from sham and PM1 treated mice were �xed in Bouin's solution, embedded in paraffin, cross-sectioned at 7 m thickness by a rotary microtome, mounted on slides, and stained by hematoxilin and eosin (HE). Some sections were mounted onto Superfrost slides and processed for the immunohistochemical detection of HO-1, as previously reported [4], using a rabbit anti-HO-1 polyclonal antibody (Santa Cruz), and the peroxidase-based Vectastain Elite ABC Kit (Vectastain Laboratories) to visualize the immunochemical reaction. Slides were observed under a Zeiss Axioplan light microscope and images taken with a ZeissAxioCam MRc5 digital camera interfaced with the Axiovision Real 4.6 soware.

Blood
Analyses. Blood of sham and PM1-treated mice was collected by intracardiac puncture. Plasma has been recovered aer two centrifugation, the �rst at 2000 g for 20 minutes and the second at 10000 g for 10 minutes at 4 ∘ C to completely remove platelets, and then submitted to sP-Selectin (Quantikine Mouse sP-selectin, R&D Systems), �brinogen (Mouse Fibrinogen Antigen assay, Molecular Innovations), PAI-1 (murine PAI-1 activity assay, Molecular Innovations), and cytokines analyses (TNF-and MIP-2; R&D Systems).

Statistical Analyses.
Results are expressed as mean ± standard error of the mean (SE). Data distribution was tested by Shapiro-Wilk test; statistical differences were tested accordingly by -test or non parametric Mann-Whitney test. Statistical differences were considered to be signi�cant at the 95% or 99% level ( or ).

Lung
Analyses. PM1 treatment induced a signi�cant reduction of Hsp70 (heat shock protein 70, a functionally related protein involved in proteins folding) and a signi�cant increase in HO-1 levels (heme oxigenase-1, a stress related protein which catalyzes heme degradation) in the lung parenchyma ( Table 2 and Figure 2(a)), while iNOS (inducible nitric oxide synthase) was unchanged (sham ± 3 ; PM1treated 2 ± 2). e immunohistochemical analyses to evidence the HO-1 expression con�rmed the activation of this antioxidant protective protein in the deep lung. HO-1 was mainly localized in AMs and in the alveolar epithelium (Figures 3(a) and 3(b)).
NF B rules the transcription of different genes, including pro-and antiapoptotic, and pro-and anti-in�ammatory ones. A signi�cant increase of its active fragment p50, as well as of the active fragment of the proapoptotic marker Caspase8-p18, was detected in the lungs of PM1-treated mice ( Table 2 and Figure 2(a)).
Cyp1B1, a cytochrome of the P450 superfamily involved in the activation of many xenobiotics and in polycyclic aromatic hydrocarbons (PAHs) metabolism, did not increase in PM1-treated mice (sham ± 2; PM1-treated 32± 7) as well as the E-selectin (sham ± 2 ; PM1-treated 33 ± 8), a cell adhesion molecule related to in�ammation. Following PM1 exposure, 24 h aer the last instillation, the histological evaluation of PM1-exposed lungs fail to disclose massive in�ammation (Figures 4(a) and 4(b)). e most signi�cant evidence in PM1 treated lungs was the ubiquitous presence in the alveolar airspace of AMs full of PM1 associated to lyses of the alveolar epithelium ( Figures  4(b) and 4(c)). ese data evidenced the active involvement of AMs in PM1 clearance and the direct cytotoxic effects elicited by PM1 on the lung alveolar epithelium, as con�rmed by LDH, ALP, and Caspase-8 analyses.

Blood Analyses.
Prothrombogenic and proin�ammatory markers were analysed within the plasma of sham and PM1-treated mice: sP-selectin, a well-known marker of the activated platelet/endothelium interface, was signi�cantly increased 24 h aer the last intratracheal instillation in PM1treated mice (

Discussion
Our previous investigations [10] disclosed that a single instillation of �ne particles in mice stimulates mild lung in�ammation. e current study extends these �ndings, showing that repeated instillations of �ne particulate matter trigger systemic adverse effect. e systemic response following repeated particle exposure could be due to a different pattern of the in�ammatory mediators released from the lung, as compared with acute exposure.
In the BALf of healthy mice, AMs are abundant (>90%) while neutrophils are rare [26]. e PM1-intratracheal instillation could facilitate the deposition of particles in the alveolar spaces, where they come in contact with AMs, the �rst cells actively engaged in the clearance of inhaled particles [27]. Many studies have demonstrated that inhaled �ne particles and aggregates of ultra�ne particles are able to burden AMs thus impairing their phagocytosis [2,28]. Such AMs in the BALf of treated mice could trigger lymphocellular in�ammatory reaction within the bronchoalveolar districts. Following particles phagocytosis, AMs usually migrate to bronchoalveolar junctions, where they tend to accumulate and aggregate [29], releasing in�ammatory mediators thus inducing a slight in�ux of neutrophils.
An increase in the number of T lymphocytes has already been demonstrated in bronchial biopsies of healthy human volunteers exposed to PM [30]; moreover, PM has been shown to drive T-cell mediated cytokine production in the BALf of treated mice [31]. Many potentially biologically active components such as endotoxin, metals, polycyclic aromatic hydrocarbons (PAHs), and ozone might activate lymphocytes in the lung of PM-treated mice [23,31]. PM1 induced changes in total and differential cells counts may be due both to a mild PMNs and Ls recruitment associated to reduced AMs migration toward the bloodstream.
In our in vivo study no signi�cant change in TNFconcentration was evident in the BALf of PM1-treated mice, while MIP-2 concentration was signi�cantly increased comparing to sham, suggesting that in�ammation is still present 24 h aer the third intratracheal instillation of PM1.
However within the lungs, PM1 failed to induce the expression of proin�ammatory adhesion molecules associated with endothelial activation, as con�rmed by the Eselectin levels basically unchanged in sham and PM1-treated mice.
Fine and ultra�ne particles have large surface area and therefore the adsorbed chemicals are largely bioavailable for redox or electrophilic chemistry [32]. It has been proposed that the proin�ammatory process induced by particles could be related to the presence of PAHs [33]. Becker et al. [34] found that also Cr, Mn, Fe, Al, Si, Ti, and Cu may be related with cytokines production. Organic chemical components and transition metals associated with PM1 may thus contribute to adverse health effects based on their ability to induce oxidative stress responsible for the lung alveolar in�ammation.
Oxidative stress and proin�ammatory cytokines are known to induce HO-1 expression in various cell types, including type II pneumocytes and AMs [35]. HO-1 acts as defence protein and its de�ciency leads to enhanced endothelial cells injury [36]: the role of HO-1 is to catabolize the heme group from the cytosol, thus generating CO, biliverdin (converted to bilirubin) and Fe 2+ ; all these products are thought to play a putative protective role against the in�ammation onset and progression [37]. HO-1 increased in PM1-treated mice in agreement with previous data related to airborne pollutant toxicity both in vivo [4,10,38] and in vitro systems [39,40], and could account for the mild ongoing lung in�ammation we found in PM1-treated mice. Due to the role of HO-1 in regulating cellular heme availability for structural and functional heme-dependent proteins [41], within lungs no change in Cyp1B1 and iNOS levels were induced by PM1. AMs in�ltrated in the lung parenchyma are positively stained for HO-1, thus suggesting that AMs in PM1-treated mice were suffering for oxidative stress due to the large burden of particles. �ithin the lungs, the pool of in�ammatory phagocytes is the most signi�cant and important cellular ROS generating system [42]; metals and organic substances adsorbed on PM surface have been related to their phagocytic oxidative burst [42].
Several transition metals adsorbed onto �ne particles have been proved to trigger the generation of reactive oxygen species, in turn able to activate NF B, one of the most important mechanisms involved in PM induced pulmonary toxicity [43]. In our investigations, p50, one of the active subunits of the NF B transcription factor, increased in PM1treated mice, in agreement with previous �ndings [10].
It has been reported [2,44] that ultra�ne particles, which are not efficiently cleared via mucociliary or macrophagemediated mechanisms, very likely may enter the epithelial cells, cause injury to the integrity of the alveolar and endothelial cells thus spreading within the circulatory system. Increased LDH and ALP activity in the BALf of PM1treated mice could be strictly related to the alveolar epithelium damage. Supporting this hypothesis and in agreement with previous investigations [4,10,14,22,41], histological analyses showed signs of alveolar cells damage within lungs of PM1-treated mice. Gerlofs-Nijland et al. [33] suggested that metals may contribute to the alveolar cell lyses and consequently to the LDH leakage in the BALf. In addition, the signi�cant increase in Caspase-8 activation found in lung parenchyma of PM1-treated mice strengthens the hypothesis of a direct cell damage and apoptosis on AMs and lung epithelial cells mediated by �ne particles [45].
It is generally known that HSPs are increased during cell stress. Surprisingly, Hsp70 levels in lung parenchyma of PM1-treated mice were signi�cantly lower than in sham. Also, Stoeger et al. [46] reported reduced Hsp70 mRNA expression aer particles instillation in BALB/c mice. Indeed, Hsp70 in the lungs is expressed by bronchial epithelium, alveolar cells and AMs [47]. HSPs may have a rapid turnover, especially during cell stress [48], and both the synthesis and chaperoning action of HSPs are energy requiring. erefore, we might speculate that an energy imbalance and the increased turnover of lung epithelial cells, as demonstrated by high ALP activity in the BALf, did not permit the synthesis of sufficient quantities of Hsp70.
Taking together, these results prove that PM1 promotes in instilled mice a mild ongoing lung in�ammation in agreement with previous �ndings [32], thus triggering prooxidative and cytotoxic effects, both on AMs and lung cells.

Effects on Mice Cardiovascular System.
Epidemiological studies provided evidences of serious health hazards linked to human exposure within highly polluted urban centres PM [49].
Growing experimental evidences suggest that inhaled smallest particles can indeed translocate into the blood systemic circulation reaching extrapulmonary organs, such as heart and brain [50]. ese adverse systemic effects might occur aer �ne or ultra�ne particles inhalation basically in the absence of symptomatic and clinically detectable lung in�ammation [32]. In our study, no variations in several in�ammation and oxidative stress markers on heart tissues of PM1-treated mice were observed. However, an increase in NF B-p50 expression has been found in heart tissue, as already described aer nanoparticles intratracheal instillation in rats [51].
Once again we observed increased activation of Caspase-8 in the hearts of PM1-treated mice, thus indicating the activation of the Caspase cascade. Cardiomyocytes apoptosis may be involved in the cardiac function impairment triggered by �ne PM [52]. ese �ndings are in agreement with the assumption that PM1 mainly exerts a direct cytotoxic effect on heart. A direct correlation has been found between �ne particles inhalation and increased �brinogen level, plasma viscosity and red blood cell count [53]. Many data indicates the adhesion of platelets to the endothelium before the development of manifest atherosclerotic lesions [54]. Furthermore, is generally accepted that platelets contribute to the �nal stages of cardiovascular diseases, thus in thrombosis and myocardial infarction [55]. Soluble P-selectin (sP-selectin) is considered a marker of an activated platelet/vasculature/blood interface, as it can be released by activated platelets as well as by activated endothelial cells [53]. Indeed, we found a signi�cant increase in sP-selectin concentration within plasma of PM1treated mice, though both �brinogen and PAI-1 concentration did not change and TNF-and MIP-2 concentration were under the kit detection limits.
Among several hemostasis and in�ammation mediators, only sP-selectin blood concentration was associated with preclinical cardiovascular risk, thus conferring to sP-selectin assay a clinical usefulness for detecting and managing high cardiovascular risk in primary prevention [56].

Conclusions
Short term exposure to PM1 induced in the lungs of BALB/c mice a mild in�ammation, still ongoing 24 h a�er the last instillation. Particles escaping phagocytosis by impaired and overloaded macrophages could then elicit their cytotoxic effect directly on alveolar cells. e inhaled PM1 could exert a progression of preexisting peripheral arterial occlusive disease sustaining the adhesion of platelets to the endothelium and considerably increasing thrombosis and myocardial infarction risks.
A better understanding of mediators and mechanisms of these processes is mandatory if strategies have to be