Differential elemental distribution of retained particles

Inhalation Toxicology, 2011; 23(8): 459–467 © 2011 Informa Healthcare USA, Inc. ISSN 0895-8378 print/ISSN 1091-7691 online DOI: 10.3109/08958378.2011.582895

RESEARCH ARTICLE

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Differential elemental distribution of retained particles along the respiratory tract Mauro A. Saieg1,2, Patricia M. Cury3, John J. Godleski4, Rebecca Stearns4, Luis G.P. Duarte3, Liz D’Agostino5, Henrique Kahn5, Emilia M. Pinto1, Thais Mauad1, Paulo H.N. Saldiva1, and Fabiola D.C. Bernardi1,2 Department of Pathology, University of São Paulo Medical School, Sao Paulo, Brazil, 2Department of Pathology, Medical Sciences School of Santa Casa de São Paulo, Sao Paulo, Brazil, 3Department of Pathology, São José do Rio Preto Medical School, Sao Paulo, Brazil, 4Department of Environmental Health, Harvard School of Public Health, Boston, MA, USA, and 5Technological Characterization Laboratory, University of São Paulo Engineering School, Sao Paulo, Brazil

1

Abstract Context: Prolonged exposure to ambient particles is associated with premature mortality due to cardio-respiratory diseases and lung cancer. The size and composition of these particles determine their toxicity, which is aggravated by their long-term retention in the lungs. Objective: To compare the elemental profile of particles retained along the bronchial tree and lymph nodes by combining laser capture microdissection (LCM) and elemental composition analysis through energy dispersive x-ray (EDX) and scanning electron microscopy (SEM). Material and methods: Twenty-four right lung middle lobes from autopsied cases were obtained from two cities with different pollution backgrounds. Lung samples were collected from three distinct sites within the lung at the time of autopsy: peribronchial tissue, peripheral parenchyma and hilar lymph nodes. Areas of potentially increased particle deposition were microdissected using LCM and analyzed for elemental composition through EDX “allied” with SEM. Results: Elemental analyses of the particles retained along the bronchial tree showed two groups of distribution: peribronchiolar or lymph node deposition. The elemental profile of peribronchial areas were significantly different between the two cities and were better discriminators of past air pollution exposure. Conclusion: Our data suggest that particle uptake varies along the bronchial tree and human lung tissue retains particles indicative of regional air pollution background. Keywords:  microdissection, lung, air pollution, microscopy, electron, scanning, spectrometry, x-ray emission

Introduction

to ambient particles are associated with premature mortality due to cardio-respiratory diseases and lung cancer (Pope et al., 2002; Eftim et al., 2008; Pope et al., 2009). Previous studies revealed that urban dwellers have particle deposition and retention in the lungs, which are associated with areas of bronchiolar fibrosis and mucus hyperplasia (Souza et al., 1998; Churg et al., 2003). Studies on the characterization of retained particles in the lungs started in the early 1970s, with identification of multiple elements of particles through energy dispersive

Ambient particulate matter (PM) is formed by a mixture of solid and liquid suspended particles, with three portions based on their aerodynamic diameter: coarse, fine and ultrafine (Lippmann et al., 1980). Size and composition are key determinants of particle toxicity, as they modulate local and systemic adverse effects associated with exposure to atmospheric pollution (Saldiva et al., 2002). Since the pioneering study of Dockery et al. (1993), it has been recognized that prolonged and repeated exposures

Address for Correspondence:  Prof. Fabiola Del Carlo Bernardi, Department of Pathology, University of São Paulo Medical School, Av. Dr. Arnaldo, 455-Cerqueira César-CEP, 01246903, São Paulo, SP-Brasil. Tel.: 005511 3061–7377; Fax: 005511 2176 1503. E-mail: fabiola@ clinicabernardi.com.br (Received 01 February 2011; revised 13 April 2011; accepted 18 April 2011)

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460  M.A. Saieg et al. x-ray analysis (EDX) while simultaneously visualizing the particles through scanning electron microscopy (SEM), using techniques such as bulk digestion or direct analysis of entire paraffin sections (Funahashi et  al., 1975; Funahashi et al., 1977; Ohshima, 1990). Since the advent of laser capture microdissection (LCM) in the mid 1990s, it has been possible to separate parts of a tissue or isolated cells through direct microscopic visualization (Simone et  al., 1998; Chimge et  al., 2007; Murray, 2007). This technique has been used in a wide variety of settings, mainly for extraction of genetic material from specific cell types in heterogeneous tissues (Simone et  al., 1998; Eltoum et  al., 2002; Chimge et  al., 2007; Cuadrado et al., 2007; Murray, 2007). Roberts et al. (2004) used LCM for the first time in an environmental study, in which airway epithelial cells from rats exposed to residual oil fly ash were microdissected for downstream molecular studies. In 2006, Liu et  al. microdissected amyloid plaques in Alzheimer’s disease and performed further elemental analysis of the microdissected material through EDX, combining these two methods for the first time (Liu, et al., 2006). Our hypothesis is that there is a differential distribution of the retained particles within the respiratory tract, due to the different mechanisms of deposition and clearance in the lung structures and to the particle characteristics. Different particle composition at different lung sites may be related to the development of specific structural abnormalities or the initiation of inflammatory responses and/or carcinogenic effects. In the present study, using LCM combined with SEM and EDX, we analyzed the elemental profile of the particles retained along the bronchial tree and lymph nodes in lungs from autopsies. We further verified whether particle uptake varied between lungs from two different air pollution environments.

Methods This study was approved by the School of Medicine of the University of São Paulo Clinical Research Ethics Board (CAPPesq-FMUSP).

Study locations São Paulo (SP) is the largest city in South America, with a population of 11 million people. Vehicular traffic is the main source of air pollution in the city, with concentrations of PM10 (PM with an aerodynamic diameter of more than 2.5 μm), PM2.5 (PM with an aerodynamic diameter of 2.5 μm or less) and nitrogen dioxide (NO2) usually found above standard levels (Annual air ­quality standards for NO2 and PM10 are 50 and 100 mg/m3, respectively).(Braga et  al., 2001; CETESB, 2007; CETESB, 2008). São José do Rio Preto (SJRP), distant 310 miles from SP, is a city with one million inhabitants. Its regional economic activity is based on cattle ranching and the agro industry, with the urban area surrounded by sugar cane plantations

and burning of sugarcane (www.planejamento.sp.gov. br). There is no annual measurement concentration of PM2.5. Measurements performed in July 2007 in that city showed that the daily concentration of PM2.5 was approximately 13 μg/m3, which is 50% lower than that found in São Paulo (CETESB, 2007). These two cities have a Death Verification Service with a large number of autopsies per year, where inhabitants that died in natural circumstances, but without a clear cause of death, undergo an autopsy to determine the cause of death.

Case selection Cases were selected from the Death Verification Services from SP and SJRP, from February to May of 2007. An interview was held with the next of kin to gather information on the clinical and epidemiological history. The inclusion criteria were as follows: individuals with ages ranging from 30 to 90 years old, never-smokers, residing for at least 30 years in the same city, no past history of heavy occupational exposure or pulmonary disease and normal lungs at autopsy.

Lung processing Lung tissue from the right middle lobe was prospectively collected from three distinct sites: central airways, peripheral lung parenchyma (including small airways) and hilar lymph nodes. Cross contamination was avoided by using non-dust gloves, changed after each sample collection. Samples were also stored in individual sterilized bags. For tissue processing and blocking, a new scalp blade was used for each sample and the work surface was wiped with 70% ethanol before and after each sample was processed. Tissue was fixed in 10% formaldehyde and paraffin embedded and 5-μm H&E sections prepared. From the tissue blocks, 10-μm sections were prepared on polyethylene teraphthlate (PET) membrane slides (Carl Zeiss, Munich, Germany) and used for LCM.

Laser capture microdissection Cases were microdissected using a PALM (Positioning and Ablation with Laser Microbeams) Microbeam IP Z microdissection system (Carl Zeiss, Munich, Germany) allied with a Zeiss Axiovert 200 microscope (Carl Zeiss, Munich, Germany) in the Molecular Biology Laboratory at the School of Medicine of the University of São Paulo. This microscope was integrated with a computer with PALM Robot Software (Carl Zeiss, Munich, Germany). Laser-cut sections were made in four distinct sites, in areas of particles uptake: subpleural region, peribronchial-central region, peribronchiolar region and hilar lymph nodes. Subpleural and peribronchiolar sections were obtained from the peripheral lung. According to Siegesmund et al. (1985), there is a correlation between particle retention and septal thickness, and when particle deposition was not present, we microdissected the

Elemental distribution of particles  461 thicker septa or the thicker adventitial area present in the sample. Dissection was confirmed by observing the corresponding negative areas in the slide (Figure 1). Laser energy was set to 70 MW/cm2 for cutting and 72 MW/cm2 for final catapulting. The catapulted microdissected fragments adhered to double-face carbon tape in a collector tube cap. A hundred dissections were performed for each site in each case, with an average total tissue area of 3 mm2 transferred to the tube cap. The tubes were stored until SEM processing.

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SEM preparation and EDX analysis The SEM analysis was performed using a Quanta 600 FEG microscope by FEI (Prague, Czech Republic) with a Silicon Drift Detector EDX, model XFlash 4030 (Bruker, Berlin, Germany), at the Technological Characterization Laboratory at the School of Engineering of the University of São Paulo. All cases were analyzed using SEM after they were sputter coated using a MED-020 coater (Bal-Tec, Liechtenstein). After the samples were platinum coated, they were stuck to an aluminum planchet using doubleface carbon tape and inserted into the SEM for combined elementary study using EDX (Figure 2). The areas of interest in the microdissected material were first chosen at a 200× magnification. The optimal magnification for particle analysis by EDX was 2000×. The electron beam of the SEM generates a number of signals when it interacts with the material. Secondary electrons are emitted during this process, collected and converted to generate a high resolution image (Funahashi et  al., 1977).

The methodology utilized to randomly select points of analysis is illustrated in Figure 3. First, two crossing lines were drawn in the two main axes of the fragments, and points along this line were chosen every 7 μm. In addition, four points of interest (one per quadrant) were also analyzed. Second, points of interest were also chosen by visualization of particles through the back scatter mode of the SEM. This methodology was repeated for each microdissected fragment until 64 points were analyzed per case. Areas containing holes or with great variation in topography were avoided. The X-rays originating from the particles were analyzed using a silicon detector, processed, and a spectrum of elements was obtained for each point of analysis through the Esprit computer software (Bruker, Berlin, Germany). The time of exposure to the beam (20 kV emission) was 60 seconds. For each spectrum, the percentages in mass for each element were obtained based on their signal strength in counts per second/electron volt. However, as samples had an irregular surface and were not polished, this data was not used. Instead, the presence or absence of a given element in each spectrum was considered for statistical purposes. Therefore, the data used for analysis was the number of points containing each element in a total of 64 points studied per case. An analysis of a microdissected stillborn lung (not exposed to ambient pollution) was also carried out as a negative control measurement. The same methodology was applied in order to validate our results and rule out a possible contamination from the H&E staining. After

Figure 1.  Sequence of events for laser capture microdissection, shown clockwise. Areas of deposition of black pigment (anthracosis) were first visualized in the H&E. These areas were chosen for cutting and catapulting. The catapulted fragments were adhered to double-face carbon tabe on the collector tube caps. Successful dissection was accessed through direct visualization of the negative areas, corresponding to the microdissected material.

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462  M.A. Saieg et al.

Figure 2.  Collector tube caps after coating, processed for scanning electron microscope analysis. Caps were placed on stubs and taken into SEM on an aluminum planchet.

collecting 64 points for elemental profiling, no elements were found (data not shown).

Statistical analysis Data are presented as medians (ranges). The comparisons between SP and SJRP samples were performed for each lung site using the Mann–Whitney U test. To test the possibility of distinguishing tissue samples from individuals living in the two areas of study based on the elemental composition of particles (cross validation), we employed a stepwise discriminant analysis, followed by a one-out approach. Briefly, the one-out approach is designed to evaluate the sensitivity of the classification done by the discriminant model to the presence of individual cases. The one-out procedure excludes a given case from the calculations and verifies the classification of the excluded case using coefficients obtained in its absence (Dawnson-Saunders, 1994). The level of significance was set to 5%. Statistical analysis was performed with the SPSS v15.0 statistical package (SPSS Inc Chicago, IL).

Results Sample characteristics Twenty-four individuals (fifteen from SP and nine from SJRP) were included in this study. Median age was 67.5 (range 38–90) years. Thirteen were male, median age 67.5, (ranged 38–77) and eleven female, median age 68 (ranged 41–90). No history of occupational exposure to metals was reported in any of the individuals. The demographic and clinical data is summarized in Table 1.

Site of retention The raw data for number of points containing elements for each lung region and study site is shown in Table 2. Only elements up to copper (Cu) are shown. Elements

Figure 3.  Example of a microdissected material visualized through scanning electron microscope (back scatter mode). 2000× magnification. Two crossing lines were drawn in the two main axis of the fragments, and points were picked every 7 μm (blue circles). Additional points of interest were analyzed, one per quadrant (yellow circles).

Table 1.  List of cases according to sex, causes of death, and time residing in the city. SP (n = 15) SJRP (n = 9) Total (n = 24) Sex (M/F) 9/6 4/5 13/11 67 (38–90) 70 (41–77) 67.5 (38–90) Age (years)* Years living in 45 (30–75) 43 (30–65) 44 (30–75) the city* Cause of death Brain edema: 2 Brain edema: 2 Brain edema: 4 MI: 3 MI: 4 MI: 7 Sepsis: 2 Sepsis: 1 Sepsis: 3 Renal Failure: 2 Renal Failure: 1 Renal Failure: 3 HM: 1 Cirrhosis: 1 HM: 1 CT: 2 CT: 2 Hepatic Failure: 1 Hepatic Failure: 1 BH: 2 BH: 2 Cirrhosis: 1 M, male; F, female; SP, São Paulo; SJRP, São José do Rio Preto; MI, myocardial infarction; HM, hypertrophic myocardiopathy; CT, cardiac tamponade; BH, brain hemorrhage. *Data expressed as median and ranges.

from antimony (Sb) to tungsten (W) were found in some rare cases, with trace signals being interpreted as individual exposure variation. An analysis of the element distribution, regardless of the study site, resulted in two main groups. The first group, which was formed by sulfur (S), chlorine (Cl), calcium (Ca) and bromine (Br), had a marked tendency for bronchiolar retention. The other group, which was formed by sodium (Na), magnesium (Mg), silicon (Si), potassium (K), titanium (Ti) and iron (Fe), was more frequently found retained in the lymph nodes. There was a significant difference for S, Ca and Br (p  =  0.001, p  =  0.021 and p  =  0.035, respectively), found significantly more often in the peribronchiolar regions, and Mg, Si, K and

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Elemental distribution of particles  463 Table 2.  Raw data of number of points containing elements, expressed in median and interquartile range by lung site and city of origin. Site Origin Na Mg Al Si P S Cl K Ca Ti Fe Br Zn Cr Mn Cu 0 0 0 0 Subpleural 0 1 62 39 0 61 59 0 18 0 4 SJRP* Median 25‡ IQR 3 6 14 25 1 27 23 7 35 11 37 19 0 0 0 0 Median 0 2 60 56 0 61 49 0 22 0 4 7 0 0 0 0 SP† IQR 4 7 8 30 0 9 26 6 27 2 23 17 0 0 0 0 Total Median 0 2 61 48 0 61 52 0 20 0 4 16 0 0 0 0 IQR 3 7 10 29 0 19 23 6 28 4 23 18 0 0 0 0 53 0 63 61§ 0 38 0 2 44|| 0 0 0 0 Peribronchiolar SJRP Median 1 3 64¶ IQR 3 6 0 16 0 1 4 2 32 2 28 23 0 0 0 0 SP Median 1 3 46 53 0 63 42 0 18 0 4 22 0 0 0 0 IQR 3 5 4 18 0 5 36 3 38 1 9 26 0 0 0 0 Total Median 1 2.5 56 53.5 0 63 58.5 0 36 0 2.5 26 0 0 0 0 IQR 3 5 2 17 0 2 18 2 37 1 9 30 0 0 0 0 32 13 0 0 1 0 Lymph node SJRP Median 4 11 64 62 0 61 48 24 18 26# IQR 19 29 4 9 0 28 25 29 39 25 29 18 0 0 1 0 SP Median 1 14 63 61 0 51 38 13 12 9 32 10 0 0 0 0 IQR 4 9 5 9 0 24 40 25 14 16 33 28 0 0 0 0 Total Median 1.5 14 63 61.5 0 53.5 45.5 13 14.5 13 32 12.5 0 0 0 0 IQR 6 22 5 9 0 23 26 26 26 25 31 23 0 0 1 0 Peribronchial SJRP Median 0 0 62 54 0 62 58** 0 9 0 0 17 0 0 0 0 IQR 5 4 20 15 0 21 20 1 14 1 2 29 0 0 0 0 0 8 0 0 0 0 SP Median 0 1 62 59 0 60 43 1 17 3†† IQR 4 7 9 15 0 18 33 3 30 4 16 13 0 0 0 0 Total Median 0 0.5 62 58.5 0 60.5 50.5 0 14 0 1.5 14.5 0 0 0 0 IQR 4 5 10 14 0 18 26 2 17 2 10 18 0 0 0 0 SJRP – São José do Rio Preto; SP – São Paulo; N – number of cases; IQR – interquartile range. *n = 9; †n = 15; ‡p  =  0.008; ¶p  =  0.006; §p  =  0.002; ||p  =  0.001; #p  =  0.006; **p  =  0.034; ††p  =  0.041.

Ti (p  =  0.013, p  =  0.021, p  =  0.001 and p  =  0.001, respectively), found significantly more often in the lymph nodes (Figure 4). When comparing elemental distribution between male and females, no difference in gender was observed, with no statistical difference observed for any of the elements analyzed.

Particle retention in SP vs. SJRP Cases from SJRP showed a greater retention of Cl (p  =  0.002), Br (p  =  0.001) and aluminum (Al) (p  =  0.006) in peribronchiolar areas, as well as Br (p  =  0.008) in subpleural areas, Ti in lymph nodes (p  =  0.006) and Cl in central areas (p  =  0.034) (Figure 5A). As for lungs from SP, Fe was more commonly found in the central areas (p  =  0.041) (Figure 5B).

Discriminant analysis A discriminant function analysis was used to determine which elemental profile at distinct lung sites could differentiate between individuals living in SP and in SJRP. Central bronchi showed a trend to greater accumulation of Al, Cu and Fe in lungs from SP, with 95.8% discrimination. When the cross-validation process (one-out procedure) was applied, the discrimination capacity was almost unaffected (91.7%), indicating that the model was robust. Subpleural, peribronchiolar and lymph nodes showed 70, 70 and 87% discrimination, r­ espectively (Figure 6).

Discussion In this study, we showed for the first time the use of LCM as a useful tool for sampling fragments for elemental analysis of retained lung particles. Using microdissected lung samples, we demonstrated that the elemental composition of the retained particles varies at different lung sites. Respiratory bronchioles and hilar lung lymph nodes had a higher frequency of particle uptake, with distinct element profiles. In addition, our data indicated that the central peribronchial areas of the lung are a better discriminator of past air pollution exposure than other lung sites. To our knowledge, this is the first study using LCM with SEM and EDX in lung pathological studies. Papers detailing the study of the lungs through SEM and EDX date back to the late 1970s and early 1980s. PM retention has classically been demonstrated in areas of anthracosis (Souza et  al., 1998; Pinkerton et  al., 2000; Churg et  al., 2003), and although several papers have detailed metal concentrations in lung tissues, including interlobar and interpersonal variations, no study has yet described the elemental composition of the anthracosis along the bronchial tree. Previous studies have used entire paraffin sections or bulk-digested tissue as methods to perform elemental analysis of retained particles in the lungs. LCM showed some advantages, as fragments analyzed were first chosen through proper histological examination, which

464  M.A. Saieg et al.

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guarantees a direct correlation to morphology and a better representation of the whole section, in a faster and more efficient way. Particle retention in the lung in areas of anthracosis associated with histological alterations in different air pollution environments was investigated by Souza et al. (1998). The authors showed that there were larger areas of anthracosis in the bronchiolar wall of nonsmokers living in an urban environment than there were in nonsmoker individuals living in a rural setting, pointing to a possible role of anthracosis as a biomarker of exposure to pollutants. Similar findings were reported by Pinkerton et  al. (2000), where a positive correlation between the degree

Figure 4.  Scheme of the bronchial tree showing the pattern of the distribution of the elements with two main groups: one with preferable peribronchiolar uptake and the other with preferable lymph node uptake. p values are shown for elements with a statistical difference between the different lung sites. P (subpleural region); B (peribronchiolar region); L (lymph node); C (peribronchial area).

of anthracosis and histological alterations (fibrosis, muscular hypertrophy and intra-luminal macrophages) was found in a series of lungs analyzed from coroner autopsies. Brauer et al. (2001) showed the relationship between inhaled and retained particles, showing that bulk-digested tissue from central portions of the lungs from Mexico City contained the same particles collected in the ambient air. However, in these studies, there was no elemental analysis of the particles. Our study describes the distribution of particles according to the elemental profile in the respiratory tract and lymph nodes. Although not quantitative, our results point to a tendency for a greater uptake of elements in peribronchiolar regions in support of previous literature (Pinkerton et al., 2000; Churg et al., 2003). Churg et al. (2003) showed a predilection for carina and segmental bronchiolar deposition of the inhaled particles in four lungs from Mexico City inhabitants. Pinkerton

Figure 5.  Scheme of the bronchial tree showing the comparison of the distribution of the elements between the two cities. (A) Elements more significantly found in Sao Jose do Rio Preto. (B) Elements more significantly found in Sao Paulo. P (subpleural region); B (peribronchiolar region); L (lymph node); C (peribronchial area); SJRP-Sao Jose do Rio Preto; SP-Sao Paulo.

Figure 6.  Percentage of discrimination of the origin of the samples (SP and SJRP) according to the elemental profile of particles deposited along the respiratory system. P (subpleural region); B (peribronchiolar region); L (lymph node); C (peribronchial area).

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Elemental distribution of particles  465 et al. (2000) found more pronounced histological alterations and deposition of black pigment in first generation bronchioles. To the best of our knowledge, this is the first study to show that the elemental composition of the particles varies along the respiratory tract. Some elements, such as S, Cl, Ca and Br, had a clear predilection for bronchiolar uptake, whereas Na, Mg, Si, K, Ti and Fe were more frequently found in the lymph nodes. This difference could be explained by different particle kinetics and clearance, with more soluble particles being found in the lymph nodes. The elements found to have a preferable bronchiolar uptake may be slowly cleared particles that could trigger local inflammatory responses and cause structural alterations that have been described at this level (Souza et al., 1998; Pinkerton et al., 2000). However, the study of the mechanisms of clearance and retention and the direct correlation of these findings to characteristics of the particles was not assessed in the present study. Further studies need to be performed to verify whether the characteristics of the particles that influence particle clearance and retention correlate with the elemental composition, validating our findings that particle uptake along the bronchial tree and lymph nodes vary in elemental composition. Assumptions can also be made to speculate on the possible variable sources of the most commonly encountered elements forming the two groups with preferable peribronchiolar or lymph node uptake. Ca, K, Mg, Fe, Na, Si and Ti may correspond mainly to resuspended soil particles, being major constituents of the crust and soil (Schauer et al., 2006). Br, S and Fe may come mainly from vehicle emission, with S and Fe commonly found as a constituent part of the Brazilian fuel, with a high incidence in gasoline and diesel. In addition, Cl is likely to have biomass burning as a potential source and Fe and Si may come from wearing of tires and breaks (Schauer et al., 2006). Our findings suggest that the more proximal regions of the bronchial tree are better discriminators of pollution exposure, being an elemental “fingerprint” of air pollution exposure. Our present study also showed that lymph nodes proved to be unsuitable for discrimination of pollution exposure, as already assigned in a study by Rogero et  al. (1994), where no difference was noted for the concentrations of most elements when the lymph nodes and entire lung samples were compared. However, they seem to carry the history of an individual’s exposition, since there was pronounced retention of Si, Ti and Fe, elements related to air pollution. In a series of studies using a technique that limits the deposition of particles to the proximal airways (Scheuch et al., 1996), a substantial fraction of particles was deposited and slowly cleared from large airways, especially the smaller particles. Another mechanism of airway retention of particles that could be related to our findings in larger airways has been proposed by previous studies. They showed that the particles deposited on top of the mucous

gel are pulled into the gel phase or even deeper into the sol phase, due to gradients of surface tension forces at the interfacial layer. Particles are then located at the base of cilia, where they appear to be immobilized and retained for prolonged periods of times, demonstrating that this site could be a final destination for a considerable amount of particles (Gehr, 1990; Gehr et al., 1996). In order to exclude a possible interference from the background constituents of lung parenchyma, statistical analysis was carried out considering only the additional points of interest in the microdissected material, chosen by visualization of particles through the back scatter mode of the SEM (data not shown). Through this approach, the results for the distribution of elements along the bronchial tree were practically the same. In our understanding, it shows that there is only a minimal contribution from the background material, what validates our study. Furthermore, an analysis of only the chosen particles could lead to a bias of profiling bigger particles, with different elemental composition. We also had the opportunity to compare the element profiles of particles retained in the lungs of individuals that lived for a long time in cities with different air pollution backgrounds. When central lung areas from SP and SJRP were compared, Fe was more frequently found in SP and Ti in SJRP. Fe and Al were more commonly found in the peribronchial areas of individuals from SP, and this might reflect exposure to vehicle emissions and resuspended traffic particles. In a study comparing lungs from two cities in Japan, Ohshima (1990) showed that the elemental composition of digested anthracotic areas had a greater concentration of Ca and nickel (Ni) in lungs from a highly polluted area, but no localization within the lung tissue was possible. A difference in gender was also noted, with higher concentrations for most of the metals found in men. Takemoto et  al. (1991) studied metal concentrations in the lungs according to gender, age, relation to emphysema and cause of death. There was a measurable increase in chromium (Cr), lead (Pb) and Ni with age, suggesting that these metals are related to pollution exposure. As for gender, Cr, Pb and Fe were mostly found in men. Our current study showed no difference between genders. A possible explanation for this finding may be the limited number of individuals enrolled in this study and the fact that 7 out of 11 female individuals had a daytime job while only four were housemaids, decreasing the percentage of time women were at home. Yet, another possible explanation for such a result may be cultural. The two above-mentioned papers are Japanese, a traditional paternalist society, and they might reflect a condition in which Japanese women have classically spent more time at home. The lack of ongoing ambient pollution monitoring for SJRP is a limitation of our study. Even though recent occasional measurements (CETESB, 2007) were performed, there is no ongoing monitoring of PM10 or PM2.5

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466  M.A. Saieg et al. or particle characterization of the ambient air pollution in that city. Furthermore, SJRP is a fast-growing city and could not be considered as a control group. This might explain some surprising results for the predominance of some elements found within these lungs. Another limitation seen in our study is the lack of sensitivity of our method, with minimal information regarding trace elements or heavy metals. Fragments adhered to the stubs were coated with platinum, part of the process for elemental analysis through SEM and EDX. The spectra corresponding to the Pt coating was easily visualized, yielding a high peak. This peak was discarded for elemental analysis and did not overlap with any of the finding elements, what corroborated to the choice of this element for coating the fragments. However, this technique is accurate for light weight elements and elements with a very low concentration in the lung tissue may not be detected.

Conclusion In summary, LCM seems to be a novel tool for sampling different areas of the lung for elemental analysis of retained particles. Laser-microdissected material is an alternative for bulk digestion of lung tissue or paraffin cut sections analysis and has proven to be useful for the determination and comparison of the elemental profile of the particles retained along the bronchial tree. Our data on the microdissected areas of increased particle uptake along the respiratory tract show that particles in the bronchioles and lymph nodes have distinct elemental compositions. The proximal bronchial tree is a main site for discrimination of pollution exposure, with an elemental “fingerprint” of air pollution. The long-term retention of different elements along the bronchial tree may be associated with the different inflammatory responses and structural alterations found within the lungs of individuals exposed to ambient particles. Therefore, further studies to assess the relationship between particle composition and the development of lung structural lesions may be helpful for improving our knowledge on the adverse effects caused by particle retention in airpolluted environments.

Acknowledgments The authors would like to thank Prof. Francis Green for his estimated help and contribution to the present study.

Declaration of interest I hereby declare that this manuscript has not been published elsewhere and it has not been submitted simultaneously for publication elsewhere. All authors have contributed to the manuscript, and aware of its submission. Prof. Paulo H N Saldiva, Prof. Thais Mauad and Prof. Patricia Cury are funded by “Fundação de Amparo

a Pesquisa do Estado de São Paulo” (FAPESP) and “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq). There are no other potential conflicts of interest.

References Braga AL, Saldiva PH, Pereira LA, Menezes JJ, Conceição GM, Lin CA, Zanobetti A, Schwartz J, Dockery DW. 2001. Health effects of air pollution exposure on children and adolescents in São Paulo, Brazil. Pediatr Pulmonol 31:106–113. Brauer M, Avila-Casado C, Fortoul TI, Vedal S, Stevens B, Churg A. 2001. Air pollution and retained particles in the lung. Environ Health Perspect 109:1039–1043. Cançado JE, Braga A, Pereira LA, Arbex MA, Saldiva PH, Santos Ude P. 2006. Clinical repercussions of exposure to atmospheric pollution. J Bras Pneumol 32 Suppl 2:S5–11. Cançado JE, Saldiva PH, Pereira LA, Lara LB, Artaxo P, Martinelli LA, Arbex MA, Zanobetti A, Braga AL. 2006. The impact of sugar caneburning emissions on the respiratory system of children and the elderly. Environ Health Perspect 114:725–729. Catalani S, De Palma G, Mangili A, Apostoli P. 2008. Metallic elements in lung tissues: Results of a meta-analysis. Acta Biomed 79 Suppl 1:52–63. CETESB-Companhia Ambiental do Estado de São Paulo. 2007. Relatório da Qualidade do Ar no Estado de São Paulo 2006. São Paulo, Brazil. Available online at http://www.cetesb.sp.gov.br. Accessed on 05 March 2009. CETESB-Companhia Ambiental do Estado de São Paulo. 2008.. “Material Particulado inalável fino (MP2,5) e grosso (MP10) na atmosfera da região metropolitana de São Paulo (2000–2006). São Paulo, Brazil. Available online at http://www.cetesb.sp.gov.br. Accessed on March 2009. Chimge NO, Ruddle F, Bayarsaihan D. 2007. Laser-assisted microdissection (LAM) in developmental biology. J Exp Zool B Mol Dev Evol 308:113–118. Churg A, Brauer M, del Carmen Avila-Casado M, Fortoul TI, Wright JL. 2003. Chronic exposure to high levels of particulate air pollution and small airway remodeling. Environ Health Perspect 111:714–718. Cuadrado E, Rosell A, Alvarez-Sabín J, Montaner J. 2007. Laser capture microdissection: A new tool for the study of cerebral ischemia. Rev Neurol 44:551–555. Dawnson-Saunders, B. 1994. Basic & Clinical Statistics. New Jersey, Prentice Hall. Dockery DW, Pope CA 3rd, Xu X, Spengler JD, Ware JH, Fay ME, Ferris BG Jr, Speizer FE. 1993. An association between air pollution and mortality in six U.S. cities. N Engl J Med 329:1753–1759. Eftim SE, Samet JM, Janes H, McDermott A, Dominici F. 2008. Fine particulate matter and mortality: A comparison of the six cities and American Cancer Society cohorts with a medicare cohort. Epidemiology 19:209–216. Eltoum IA, Siegal GP, Frost AR. 2002. Microdissection of histologic sections: Past, present, and future. Adv Anat Pathol 9:316– 322. Funahashi A, Pintar K, Siegesmund KA. 1975. Identification of foreign material in lung by energy dispersive x-ray analysis. A new approach to silicosis. Arch Environ Health 30:285–289. Funahashi A, Siegesmund KA, Dragen RF, Pintar K. 1977. Energy dispersive x-ray analysis in the study of pneumoconiosis. Br J Ind Med 34:95–101. Gehr P, Green FH, Geiser M, Im Hof V, Lee MM, Schürch S. 1996. Airway surfactant, a primary defense barrier: Mechanical and immunological aspects. J Aerosol Med 9:163–181. Gehr P, Schurch S, Berthiaume Y, Im Hof V, Geiser M. 1990. Particle retention in airways by surfactant. J Aerosol Med 3: 27–43.

Inhalation Toxicology Downloaded from informahealthcare.com by St. Jude Children's Research Hospital on 11/07/13 For personal use only.

Elemental distribution of particles  467 Governo do Estado de São Paulo (São Paulo State Government). 2007. Economy Secretariat Website. Available online at http://www. planejamento.sp.gov.br. Accessed on 16 September 2009. Lippmann M, Yeates DB, Albert RE. 1980. Deposition, retention, and clearance of inhaled particles. Br J Ind Med 37:337–362. Liu G, Huang W, Moir RD, Vanderburg CR, Lai B, Peng Z, Tanzi RE, Rogers JT, Huang X. 2006. Metal exposure and Alzheimer’s pathogenesis. J Struct Biol 155:45–51. Murray GI. 2007. An overview of laser microdissection technologies. Acta Histochem 109:171–176. Ohshima S. 1990. Studies on pulmonary anthracosis. With special reference to the mineral constitution of intrapulmonary particulate pollutants in the human lung. Acta Pathol Jpn 40:41–49. Pignatello JJ, Cohen SZ. 1990. Environmental chemistry of ethylene dibromide in soil and ground water. Rev Environ Contam Toxicol 112:1–47. Pinkerton KE, Green FH, Saiki C, Vallyathan V, Plopper CG, Gopal V, Hung D, Bahne EB, Lin SS, Ménache MG, Schenker MB. 2000. Distribution of particulate matter and tissue remodeling in the human lung. Environ Health Perspect 108:1063–1069. Pope CA 3rd, Burnett RT, Thun MJ, Calle EE, Krewski D, Ito K, Thurston GD. 2002. Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution. JAMA 287:1132–1141. Pope CA 3rd, Ezzati M, Dockery DW. 2009. Fine-particulate air pollution and life expectancy in the United States. N Engl J Med 360:376–386. Roberts E, Charboneau L, Espina V, Liotta L, Petricoin E, Dreher K. 2004. Application of laser capture microdissection and protein microarray technologies in the molecular analysis of airway injury following pollution particle exposure. J Toxicol Environ Health Part A 67:851–861.

Rogero SO, Saiki M, Saldiva PH, Daliberto ML. 1994. Determination of trace elements in human lung samples. Biol Trace Elem Res 43-45:489–496. Saldiva PH, Clarke RW, Coull BA, Stearns RC, Lawrence J, Murthy GG, Diaz E, Koutrakis P, Suh H, Tsuda A, Godleski JJ. 2002. Lung inflammation induced by concentrated ambient air particles is related to particle composition. Am J Respir Crit Care Med 165:1610–1617. Schauer JJ, Lough GC, Shafer MM, Christensen WF, Arndt MF, DeMinter JT, Park JS. 2006. Characterization of metals emitted from motor vehicles. Res Rep Health Eff Inst. 133:1–76; discussion 77-88. Scheuch G, Stahlhofen W, Heyder J. 1996. An approach to deposition and clearance measurements in human airways. J Aerosol Med 9:35–41. Siegesmund KA, Funahashi A, Yorde DE. 1985. Morphometric and elemental microanalytical studies of human lung in health and disease. Br J Ind Med 42:36–42. Simone NL, Bonner RF, Gillespie JW, Emmert-Buck MR, Liotta LA. 1998. Laser-capture microdissection: Opening the microscopic frontier to molecular analysis. Trends Genet 14:272–276. Socko R, Kupczewska-Dobecka M. 2007. Is dichloromethane an occupational carcinogen? Med Pr 58:143–153. Souza MB, Saldiva PH, Pope CA 3rd, Capelozzi VL. 1998. Respiratory changes due to long-term exposure to urban levels of air pollution: A histopathologic study in humans. Chest 113:1312– 1318. Takemoto K, Kawai H, Kuwahara T, Nishina M, Adachi S. 1991. Metal concentrations in human lung tissue, with special reference to age, sex, cause of death, emphysema and contamination of lung tissue. Int Arch Occup Environ Health 62:579–586.