Pleural anthracosis as an indicator of lifetime exposure to urban air pollution: An autopsy-based study in Sao Paulo

Environmental Research 173 (2019) 23–32

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Pleural anthracosis as an indicator of lifetime exposure to urban air pollution: An autopsy-based study in Sao Paulo

T

Ana Paula Cremasco Takanoa,f, Lisie Tocci Justoa, Nathalia Villa dos Santosa, Mônica Valeria Marquezinia, Paulo Afonso de Andréa, Francisco Marcelo Monteiro da Rochae, Carlos Augusto Pasqualuccia, Lígia Vizeu Barrozoc, Julio M. Singerb, ⁎ Carmen Diva Saldiva De Andréb, Paulo Hilário Nascimento Saldivaa,d, , Mariana Matera Verasa a

Universidade de Sao Paulo Medical School (FMUSP), Sao Paulo, Brazil Institute of Mathematics and Statistics, University of Sao Paulo, Sao Paulo, Brazil Department of Geography, School of Philosophy, Literature and Human Sciences, University of Sao Paulo, Sao Paulo, Brazil d Institute of Advanced Studies, University of Sao Paulo (IEA-USP), Sao Paulo, Brazil e Escola Paulista de Economia e Negócios (EPPEN), Federal University of Sao Paulo (UNIFESP), Sao Paulo, Brazil f Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Anthracosis Air pollution Lung Exposure assessment

Many studies have been conducted to evaluate the association between air pollution and adverse health effects using a wide variety of methods to assess exposure. However, the assessment of individual long-term exposure to ambient air pollution is a challenging task and has not been evaluated in a large autopsy study. Our goal was to investigate whether exposure to urban air pollution is associated to the degree of lung anthracosis, considering modifying factors such as personal habits, mobility patterns and occupational activities. We conducted a study in Sao Paulo, Brazil from February 2017 to June 2018, combining epidemiological, spatial analysis and autopsybased approaches. Information about residential address, socio-demographic details, occupation, smoking status, time of residence in the city and time spent commuting was collected via questionnaires applied to the next-ofkin. Images of the pleura surface from upper and lower lobes were used to quantify anthracosis in the lungs. We used multiple regression models to assess the association between the amount of carbon deposits in human lungs, measured by the fraction of pleural anthracosis (FA), and potential explanatory variables. We analyzed 413 cases and our data showed that for each additional hour spent in daily commuting, the ratio FA/(1-FA) is multiplied by 1.05 (95% confidence interval: [1.02; 1.08]). The estimated coefficient for daily hours spent in traffic was not considerably affected by the inclusion of socio-demographic variables and smoking habits. We estimate a tobacco equivalent dose of 5 cigarettes per day in a city where annual PM2.5 concentration oscillates around 25 μg/m3. Pleural anthracosis is a potential index of lifetime exposure to traffic-derived air pollution.

1. Introduction

The usual assessment of the exposure to air pollutants is based on retrospective data from fixed monitoring networks from which annual or monthly average concentrations are derived (Lepeule et al., 2014b; Lipsett et al., 2006). This provides data for points in space and can overor underestimate individual exposure because it does not consider individual activities. In addition to ambient effects, individual exposure to air pollutants is influenced and determined by many factors, such as time spent outdoors, indoor air pollutant levels, location, daily activities, mobility, mode of transportation, occupation and personal habits (Buonanno et al., 2014). To overcome this, different approaches have

Ambient air pollution has been consistently associated with shortand long-term adverse health effects and represents one of the major environmental health risks in urban areas (Fajersztajn et al., 2017; WHO, 2016). Most evidence of the impacts posed by air pollution on human health comes from epidemiological studies. However, these studies are challenged with respect to the assessment of individual and lifetime exposure (Brauer et al., 2002; Bravo et al., 2012; Zeger et al., 2000).

⁎ Correspondence to: Laboratory of Environmental Air Pollution, Department of Pathology of the University of Sao Paulo Medical School, Av. Dr. Arnaldo, 455, Cerqueira Cesar, 01246903 Sao Paulo, SP, Brazil. E-mail address: [email protected] (P.H.N. Saldiva).

https://doi.org/10.1016/j.envres.2019.03.006 Received 6 November 2018; Received in revised form 1 March 2019; Accepted 2 March 2019 Available online 04 March 2019 0013-9351/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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Fig. 1. Study flow chart. The study associated clinical assessment steps with pathological procedures to estimate an autopsy-based assessment of urban air pollution exposure. The study had considered 430 cases at the beginning of the assessed steps, but only 413 cases were finally evaluated after exclusions in step 4 of clinical assessment.

and occupational activities. Megacities, especially in developing countries, are particularly important for the study of the adverse health effects of air pollution because they combine a large exposed population and pollutant levels exceeding WHO recommended standards. In fact, megacities are a global reality and their number is increasing steadily along with the number of vehicles, especially automobiles, which are responsible for about two thirds of the air pollution in these areas (Bhandarkar, 2013). Sao Paulo fits perfectly in the aforementioned scenario. The municipality of Sao Paulo, with more than 12 million inhabitants, is among the 10 most populous in the world and is the first in Latin America. Traffic in Sao Paulo represents the main source of air pollution with approximately 75% of primary particulate matter emitted by vehicles (CETESB, 2017). Black carbon, which is the major pollutant related to anthracosis, is almost exclusively produced by vehicle and tail-pipe emissions, with equal contribution of light and heavy-duty-vehicles (Brito et al., 2018). In Sao Paulo, environmental and health data are available, air pollution levels are high enough to impose significant health effects to population characterized by great social and demographic variability. Furthermore, Sao Paulo has unique characteristics that provide the perfect scenario to develop an autopsy-based study. It has a large autopsy service that performs about 15,000 compulsory cases per year. We take this as an advantage to evaluate the association between individual cumulative exposure to traffic related air pollution and the amount of carbon deposits in human lungs based on autopsied cases. Our hypothesis is that the quantity of pleural anthracosis could be an indicator of the cumulative exposure to traffic air pollution.

been used, such as numerical models (Korek et al., 2016; Lepeule et al., 2014a), satellite imaging (Anderson et al., 2012), personal monitors (Okokon et al., 2017; Santos et al., 2016) or a combination thereof (Eeftens et al., 2012; Huck et al., 2017; Pigliautile and Pisello, 2018). However, the cost and technology necessary to conduct such exposure assessments can be restrictive. More precise assessment of individual exposure is crucial for the establishment of causal relationships, for tracking and monitoring air pollution as well as for local policy planning. Recent studies on this topic point out the need to improve the methods to assess personal exposure more accurately (Park and Kwan, 2017), giving citizens the opportunity and knowledge to change behavior and consequently, to reduce their individual exposure. Anthracosis (anthrac- meaning coal, carbon + osis meaning condition) due to inhalation of coal dust results in black deposits of carbon, particularly in the lungs. Pulmonary anthracosis, caused by repeated inhalation of particulate air pollutants, smoke or coal dust particles (Mirsadraee, 2014; Saieg et al., 2011), can be macroscopically identified by the presence of black patches on the lungs, accompanied by pleural anthracosis. These carbon particles deposited in the respiratory system, as well as trace elements adsorbed in its graphitic core or surface (Stuart, 1984; Tsuda et al., 2013; Yeh et al., 1976), may be retained in the pleural region and lymph nodes for a long period. In 1963, Zeidberg and Prindle suggested that pleural anthracosis could serve as an index of air pollution exposure. In their study, histopathological sections of autopsied lungs from Nashville residents (a city with coal derived pollution) were classified according to the degree of anthracosis. The results showed that the amount deposited in the lungs had a positive association with the duration of exposure (time living in the city), with the caveat that smoking habits were not considered. The amount of black carbon accumulated in airway macrophages is associated with regional variations of ambient pollution particle concentration in children (Kulkarni et al., 2006) and adults (Bai et al., 2018) and thus anthracosis may be considered as a proxy variable of long-term black carbon accumulation in pulmonary parenchyma. Considering further robust evidences (Brauer et al., 2001; Tsuda et al., 2013) that human lungs retain ambient particles, we decided to move forward in this topic and investigate whether exposure to urban air pollution is associated to the degree of pleural anthracosis, taking into account modifying factors such as personal habits, mobility patterns

2. Methods This study is part of the MetroHealth subproject of a project entitled The Use of Modern Autopsy Techniques to Investigate Human Diseases (MODAU) and was approved by the Research Ethics Committee of the University of Sao Paulo (number 537.195). MetroHealth addresses the effect of different aspects of life in megacities on the pathogenesis of human diseases combining epidemiological, spatial analysis and autopsy-based approaches. We present a new strategy to quantify the accumulated lifetime exposure to urban air pollution by measuring anthracosis in the lungs. A flow chart of the study design is depicted in Fig. 1. 24

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former smoker), environmental tobacco smoking (yes or no), number of cigarettes smoked per day and years of active smoking. Based on these variables, the smoking amount in pack years was computed.

2.1. Study population and post mortem evaluation The study was conducted at the Death Verification Service of Sao Paulo (SVOC) from February 2017 to June 2018. Up to 3 cases per day were enrolled from Mondays to Fridays, from 8 a.m. to 4 p.m. In Brazil, autopsies are compulsory for all undefined natural deaths. The SVOC is responsible for these autopsies, defining the cause of death of approximately 20% of the natural deaths occurred in Sao Paulo (Ministry of Health, 2018). As a routine procedure, the corpses of the deceased are transferred to the SVOC and the body claimed by a relative (next-ofkin “NOK”) who signs a term allowing the autopsy procedure. In our study, following this procedure, a trained interviewer invited the NOK to participate, explaining its purpose and relevance. Upon acceptance, the relative was taken to a private room where an informed consent form was voluntarily signed, authorizing the collection of lung images and tissue samples. In the sequence, the relative was asked to complete a questionnaire with information about the deceased, including previous health conditions, residential address, socio-demographic details, life habits, smoking status, occupation, time of residence in Sao Paulo and time spent commuting. Inclusion criteria for the study were: i) to have died of natural death; ii) age equal or greater than 18 years; iii) be living in Sao Paulo at the time of death; iv) have one close relative to provide reliable and complete information during the interview; and v) present no macroscopic alterations of the lungs, such as excessive fibrosis or very dark red-purple coloration that interfere anthracosis measurement.

2.5. Exposure to traffic Occupation, number of years of residence in Sao Paulo, number of years in the current address and the number of hours spent commuting between home and work were obtained via the questionnaire to assess individual exposure scenarios. For individuals whose occupational activity imposed higher exposure to traffic (taxi drivers, truck drivers, mailmen, etc.), 8 h were added to the daily commuting time. The proportion of the lifespan living in Sao Paulo was computed as the number of years of residence in Sao Paulo/age. We also considered traffic intensity at each address as a modifier of exposure. The addresses of all the deceased were geocoded using the MapInfo Professional 7.0 software and the georeferenced street cartographic base with street classification relatively to traffic intensity according to the city traffic authority (CET - Companhia de Engenharia de Tráfego, 2013). This classification is based on street characteristics and the importance of the traffic flow. It consists of four categories: Express, Arterial, Collector or Local (in decreasing order of traffic intensity). We computed the distances from the deceased residence to the closest express, arterial or collector street and the minimum value was considered as a measure of the distance from the residence to closest street with major traffic intensity. Using ArcGis 10.3, with the shapefile file of streets, a raster file was generated around each recorded address and the values of street density given by the total number of linear meters of streets divided by the area (m/m2) in a buffer distance of 100 m were extracted.

2.2. Anthracosis index During the autopsy, lungs were removed and the excess of blood from the pleural surface was cleaned. Then a 10 cm Petri dish was superimposed on the anterior surfaces of the upper and lower lobes of both lungs to flatten the observation area and photographs of the pleura surface were taken with a high-resolution camera (Canon Power Shot SX400 IS). Using these 4 images from the flattened surface of the lungs, the fraction of anthracosis was estimated by the point counting method (Howard and Reed, 1998) with the aid of the ImageJ software (https:// imagej.nih.gov/ij/docs/intro.html) to generate the point test system. For each of the four lobes, points falling on black patches (BP) and in the clean pleura (NBP) were counted and the fraction of anthracosis (FA) for each lung lobe determined as: FA = number of BP / (number of BP + number of NBP). A detailed and schematic illustration of the protocol can be found in the Supplementary material (Fig. S1). Then, we computed the mean of the FA for the upper lobes as well as the mean for the lower lobes values.

2.6. Socioeconomic index We considered a socioeconomic index built by Barrozo et al. (2017) to help public health practitioners identify vulnerable conditions related to health in the Municipality of Sao Paulo. The index is not obtained with individual information of the deceased; it is based on the sample areas of the 2010 demographic census and involves 27 variables related to income, poverty, wealth, education, social and material deprivation and cultural aspects. The variables with highest weight in the index are dweller density by room, percentage of people with no instruction or incomplete basic level and percentage of people whose ethnicity is black, mixed race or indigenous. Its values range from -1 to 1 and high values indicate better socioeconomic conditions. 2.7. Statistical analysis

2.3. Histopathological evaluation

We used multiple regression models to assess the association between the amount of carbon deposits in human lungs as measured by the FA and daily commuting, controlling by lobe position (upper or lower), smoking amount, environmental tobacco smoke, age, years living in Sao Paulo, socioeconomic index, street density in a buffer of 100 m and distance from the deceased residence to the closest street with major traffic intensity. The models were chosen so that predicted FA lies between 0 and 1. For such purposes, we considered multivariate Gaussian nonlinear models with an unstructured covariance matrix fitted by restricted maximum likelihood. The unstructured covariance matrix accounts for possible within lung FA correlations. The logit of the FA, i.e., log [(FA)/(1-FA)], was used as the link function so that the interpretation of the coefficients of the explanatory variables is similar to that considered in logistic regression models, i.e., as logarithms of odds ratios. For details, see Davidian and Giltinan (1995). The analysis strategy started by fitting an initial model in which hours spent in traffic was the only explanatory variable and proceeded by the subsequent inclusion of the remaining explanatory variables to evaluate the sensitivity of the model coefficient for hours spent in

Anthracotic regions of the lung surfaces were assessed microscopically to qualitatively characterize tissue changes and a subset of the samples was used to quantify particle deposition. For this, 1 cm³ samples of macroscopically photographed regions were also collected for histopathological and histochemical analysis. These samples were fixed for 24 h in 4% formalin and routinely processed for histology. Three sections perpendicular to the pleural surface (5 μm thickness) from each paraffin block were obtained and stained with Hematoxylin and Eosin (H&E), with Picrosirius Red for fibrosis analysis and with Perls´ Prussian Blue for iron pigment detection. Images were visualized and captured using a Nikon Eclipse E200 microscope coupled to the camera. 2.4. Exposure to tobacco smoke Multiple potential predictors related to the use of tobacco were included in the questionnaire: smoking habit (non-smoker, smoker or 25

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traffic. The robustness of the results was evaluated by fitting beta regression models with a logit link function to the average FA in each lung (Cribari-Neto and Zeileis, 2010; Ferrari and Cribari-Neto, 2004). Given that the average FA in the lobes evaluated in the same lung could be a biased index of anthracosis in cases with missing measurements, we repeated the analysis using only the cases with all four measurements. Diagnostic tools for nonlinear models as described in Neter et al. (2005) and for beta regression models (Ferrari and Cribari-Neto, 2004) were employed to check the appropriateness of the model assumptions. The functions gnls and betareg available in the R software (version 3.4.3) were used to fit the models.

some lobes inside the thoracic cavity during the lung removal in the autopsy procedure. Cases without the complete lung lobes were controlled by statistical modeling. The mean FA was 0.23 (standard deviation = 0.16) for the upper lobes and 0.18 (standard deviation = 0.14) for the lower lobes (Table 2). The response variable was the mean of the left and right anthracosis index for either lower or upper lobes.

3.3. Histopathological evaluation Although the focus of this study was the quantitative evaluation of pleural anthracosis at the macroscopic level, we also collected lung samples of all cases, which permitted the complementary description of qualitative histopathological aspects observed in histological preparations. Histopathological and histochemical analyses of lungs from both smokers and non-smokers evidenced the presence of respiratory bronchiolitis, characterized by fibrosis and narrowing of the walls of terminal and respiratory bronchioles, rupture of alveolar walls at centriacinar region. Particles were accumulated mostly in the peribronchiolar connective tissue, as well as around intra-acinar branches of pulmonary artery. To a lesser extent, particles were also seen, within intraluminal alveolar macrophages and interstitial spaces. In the pleural region, the black spots were mainly associated with the subpleural lymphatic system. The black pleural areas exhibited also close relation to the connective tissue of the interlobular spaces, providing anatomical evidences of recruitment of all pulmonary lymphatic systems (bronchial, vascular, interlobular and pleural) in the processes of clearing inhaled black carbon. In fact, the histological evidence of increased function of all pulmonary lymphatics denote that inhaled carbon reaches all pulmonary compartments. In such context, our data (in 36 subjects) indicate that the amount of intrapulmonary carbon spots are significantly associated with pleural anthracosis (Fig. S2). Carbon particles elicit a chronic inflammatory response, characterized by recruitment of inflammatory cells (mostly macrophages and lymphocytes) and distortion of normal pulmonary and pleural histoarchitecture by fibrosis. The histological alterations caused by tobacco or by air pollution are quite similar, with the exception of the iron content for tobaccoexposed individuals. Iron is a normal component of the so-called smoker´s pigment (Pappas, 2011), probably due to the alterations of iron alveolar homeostasis – increased extravasation of ferritin to alveolar lumen – induced by tobacco (Ghio et al., 2008). Representative pictures are depicted in Fig. 5. Additionally, comparative histopathological aspects between individuals with low and high exposure to ambient levels of air pollution are specified in Fig. 6.

3. Results 3.1. Study population characteristics The studied sample included the first 430 individuals that met the inclusion criteria. For 17 cases, we could neither geocode their addresses nor have traffic information near their residences. These cases were not included in the analysis. The sample characteristics are summarized in Table 1. The age range observed in the sample is similar to that of the population of individuals older than 18 years who died from natural deaths in the city of Sao Paulo in 2017 (Ministry of Health, 2018), and the values of the sample socioeconomic index varies from -1 to 1. The spatial distribution of residences as well as of the socioeconomic index are depicted in Fig. 2. The large gaps in the map are due to the presence of green areas and reservoirs (Fig. S3). The cases are well distributed throughout the city. An analysis of the lifespan spent in Sao Paulo showed that 32% of the cases lived in the region for their entire lives and 50% of the cases lived there for at least 75% of their lives. Furthermore, only 20% of the cases lived in Sao Paulo for less than 50% of their lifespan. Details are presented in Fig. 3. 3.2. Anthracosis index We focused on macroscopic observation and quantification of anthracosis to estimate individual chronic exposure to urban ambient particles taking into account concomitant exposure to cigarette smoke. Fig. 4 shows representative aspects of carbon deposition in the pleural surface, indicating that smokers and non-smokers do exhibit visible areas of anthracosis deposition. For some cases it was not possible to photograph all the four lobes (n = 53), which means that for some cases we have 3 or 2 pictures. This was due to the pleural adhesion of Table 1 Characteristics of 413 cases included in the study. Variable

Mean

Sd

Minimum

Median

Maximum

Iqr

Age (years) Male Socio-economic index Years living in Sao Paulo Proportion of lifespan living in Sao Paulo Smoker or former smoker Smoking amount (pack years) Environmental tobacco smoke Daily commuting (hours) Outdoor work exposed to traffic Work at home Distance from home to the closest express, arterial or collector streets (m) Street density in a buffer of 100 m around home (m/m2) Death by diseases of the circulatory system Death by diseases of the respiratory system Death by diseases of the digestive system Death by other causes

68.5

15.3

19

69.0

110

23

−0.24 48.3 0.71

0.36 21.7 0.29

−1.0 0.3 0.003

−0.32 50.0 0.75

1.0 103 1

0.47 24 0.43

23.6

39.3

0

0.0

256

38

1.8

2.5

0

1.0

11

2

181.8 0.02

209.4 0.01

Sd: standard deviation; Iqr: interquartile range. 26

0.2 0.00

121.6 0.02

1964 0.04

N

%

221

53.5

197

47.7

143

34.6

35 125

8.5 30.3

252 60 29 72

61.0 14.5 7.0 17.4

194.8 0.01

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Fig. 2. Spatial distribution of residences and the socioeconomic index (SES index) of the neighborhoods of necropsied individuals included in the study.

between time spent in traffic and the amount of anthracosis in the pleura (FA). The estimated coefficient of daily hours spent in traffic was not considerably affected by the inclusion of socio-demographic variables and smoking habits. Similar conclusions were obtained when fitting of the beta regression models, both when all the 413 cases were considered (Table S1) and when the 360 cases with observed values of anthracosis in the four lobes were included in the analysis (Table S2). The coefficient of hours spent in traffic in model 5 in Table 3 implies that, for each one hour increase in daily commuting, the FA / (1-FA) ratio is multiplied by exp (0.047) = 1.05 (95% confidence interval: [1.02; 1.08]), that is, the ratio increases by 5%. The ratio between the coefficients of daily hours spent in traffic and smoking amount is equal to 9.4, which implies that one hour of daily commuting is equivalent to 9.4 packs year. Considering that the average time of tobacco use for smokers is 40 years, the models suggest that one hour in daily commuting during the active life of an inhabitant of Sao Paulo is equivalent, in terms of anthracosis, to smoking 5 cigarettes per day.

Fig. 3. Percentile chart of the proportion of lifespan living in Sao Paulo.

3.4. Association between anthracosis index and individual characteristics

4. Discussion

The results of the macroscopic assessment of anthracosis are summarized in Table 2. For descriptive purposes, the smoking amount categories were: non-smokers, smoking less than the median smoking amount (40 pack years) and more than that. Daily commuting was categorized as: no daily commuting, daily commuting less than 2 h and daily commuting 2 h or more. The observed FA means are higher in the upper lobe than in the lower lobe and tend to increase with increasing smoking amount and with commuting time. The results obtained by fitting the nonlinear regression models with incremental inclusion of controlling variables are summarized in Table 3. Briefly, our results show that there is a significant association

Our results indicate that the proportion of anthracosis in the pleural surface of the lungs is positively associated with exposure to traffic derived air pollution and age, suggesting a time-dependent pattern. They reinforce previous histological findings that carbon deposition in human lungs increases with age (Zeidberg and Prindle, 1963) and that, in urban areas, it is highly associated with exposure to traffic sources (Brauer et al., 2001). Carbon deposition was evaluated by macroscopic approach for two main reasons: first, we considered that a macroscopic approach integrates a much larger fraction of the lung than a microscopic analysis and pleural anthracosis is significantly associated with intrapulmonary 27

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Fig. 4. Macroscopic view of anthracosis. Representative lung images from upper lobe with areas of black carbon deposition in the pleural surface. A) smoker (50 pack years), 65y old, one daily hour spent in traffic; B) nonsmoker, 63-y old, four daily hours spent in traffic. Both individuals were male and Sao Paulo dweller for 50 years. FA: fraction of anthracosis.

Table 2 Sample sizes, mean fraction and standard deviation of the mean pleural anthracosis in the upper and lower lobes disaggregated by the categories of daily commuting and smoking amount. Smoking amount Non-smokers

Upper lobe

Lower lobe

< 40 pack years

> = 40 pack years

Total

Daily commuting

N

Mean

Sd

N

Mean

Sd

N

Mean

Sd

N

Mean

Sd

No commuting Less than 2 h 2 h or more Total No commuting Less than 2 h 2 h or more Total

81 63 72 216 81 63 72 216

0.18 0.18 0.20 0.19 0.16 0.13 0.16 0.15

0.14 0.13 0.13 0.13 0.14 0.11 0.12 0.13

22 39 36 97 21 39 36 96

0.22 0.25 0.28 0.26 0.18 0.18 0.22 0.20

0.16 0.14 0.16 0.15 0.18 0.12 0.15 0.14

22 37 41 100 22 37 41 100

0.25 0.34 0.34 0.32 0.18 0.23 0.26 0.23

0.17 0.20 0.18 0.19 0.17 0.18 0.15 0.17

125 139 149 413 124 139 149 412

0.20 0.24 0.26 0.23 0.17 0.17 0.20 0.18

0.15 0.17 0.17 0.16 0.15 0.14 0.14 0.14

Sd: Standard deviation. The response variable was the mean of the left and right anthracosis indices obtained in either upper or lower lobes.

major sources; they can be emitted directly from primary sources (traffic, industry), they can be formed by photochemical reactions of the primary pollutants in the atmosphere or by re-suspension of particles present in the soil (Brook, 2008; Karagulian et al., 2015). The inhalable particles range from 10 μm to few nanometers and, depending on the size, they are categorized as coarse (2.5–10 μm), fine (0.1–2.5 μm) and ultrafine particles (0.01–0,1 μm). Their concentrations as well as size distribution vary in time and space (Hussein et al., 2004; Oliveira et al., 2009). Size is the most important factor because it determines both the behavior and the deposition mechanism of the particles (Hinds, 1999). Once particles enter our respiratory system, depending on their characteristics, they can be deposited or removed by different clearance pathways. Larger particles tend to deposit in the airways and are cleared; the smaller ones deposit in the alveolar region; insoluble particles can be translocated and accumulate in the tracheobronchial lymph nodes and pleura (Karakoti et al., 2006; Tsuda et al., 2013). The lung clearance mechanisms have limitations because not all the inhaled and deposited particles can be removed by macrophages or by the mucociliary escalator. Furthermore, exposure to high levels of particulate matter overwhelms the defense mechanisms impairing their efficiency. Clearance mechanisms of particles within the lower regions of the lungs are mostly mediated by macrophages (Oberdörster et al., 2005). This mechanism is affected by high dose exposure, diminishing its efficiency due to macrophage particle overload (Hinds, 1999). Although we know that particle size, breathing pattern and the presence of respiratory diseases (Brown et al., 2002) are factors that influence deposition within the lungs, it is important to note that we did not exclude individuals who died by respiratory diseases (14.5% of all cases), due to the absence of pleural macroscopic alterations.

anthracosis (as evaluated on a smaller subset of subjects included in the study and showed in Fig. S2). The observed saturation for higher FA could be explained by a sampling bias due to the reduced representative fraction of the surface evaluated using histology, associated to efficiency of pulmonary clearance in elderly and smokers or differences in particles regional deposition (Carvalho et al., 2011; Geiser and Kreyling, 2010). Second, since the aim of our study was to conduct the anthracosis measurements in a large sample, macroscopic pleural measurements are less expensive, less time-consuming and could be easily reproduced in other settings that have autopsy services, even in developing countries. Using lung samples from autopsies but with a different methodology, Brauer et al. (2001) have investigated if lung parenchymal particle burden could be indicative of individual air pollution exposure. Their results suggested that there is a direct association between increased ambient levels of exposure and higher particle retention in the lungs. Similarly, carbon content in airway macrophages is associated to the total particulate burden in the lung (Kulkarni et al., 2006). A previous study conducted by Padovan et al. (2017) did not find an association between exposure to traffic and carbonaceous deposition in the lung surface. This can be explained by the incomplete assessment of individual exposure, small number of cases and biased quantification of the fractional area of anthracosis in the pleural surface. In their study, determinant variables for the characterization of exposure, like passive smoking, occupation and commuting time were not considered. Moreover, the quantification of anthracosis was limited to a single lung lobe, and it is well known that deposition of particles in the lung has regional differences (Mitchev et al., 2002). Aerosols and particulate matter present in urban air have three 28

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Fig. 5. Representative microscopic appearance of anthracosis (black patches) in the upper lung lobe. A, B and C are images from upper lobe of a 78-y old woman (having always resided in Sao Paulo), non-smoker, housewife, and have died by myocardial infarction. Panel D, E and F represent images from the upper lobe of a 67y old woman (having resided in Sao Paulo for only 1 year), smoker with 1.5 pack-years, housewife and have died by pulmonary thromboembolism. Top images were stained with Perls´ Prussian Blue evidencing the iron deposition (blue color) characterizing black particles derived mainly from exposure to tobacco smoke. Middle images represent Picrosirius Red staining showing the black particles embedded within fibrous areas (red staining) near centriacinar regions (arrow). Bottom images were stained with H&E and highlight the deposition of black particles near perivascular areas (arrow), thickening of lung septa (arrowhead) and presence of inflammatory cells (*). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

identification of the mechanism responsible for the high deposition efficiency of traffic derived particles. Conceivably, the much higher concentration of black carbon in tobacco smoke may favor aggregation of inhaled particles during their transit along the airways, thus favoring deposition in more proximal segments of the respiratory tract, facilitating their removal by pulmonary clearance mechanisms, In this context, our results may help epidemiologists in the process of selecting more robust estimators of particle exposure, or, alternatively, of attributing different weights when designing population based epidemiologic studies. In a short memo, Muller and Muller (2013) pointed that the average of PM2.5 in Beijing over the year is about 85 μg/m3, which is equivalent to about 4 cigarettes per day. They used a rule that one cigarette per day is the rough equivalent to a PM2.5 level of 22 μg/m3. Van der Zee et al. (2016) showed that health risks of exposure to 10 μg/m3 PM2.5 is equivalent to 5.5 passively smoked cigarettes per day. In our results, in a city with a PM2.5 annual mean concentration around 25 μg/m3, we estimated a tobacco equivalent dose of 5 cigarettes per day by comparing the relative contribution of tobacco and outdoor pollution to pulmonary anthracosis. It is probable that traffic exposure as well as individual factors, such as aging leading to changes in pulmonary architecture, comorbidities and alterations of alveolar clearance functioning (Hanania et al., 2011; Skloot, 2017), may increase individual dose of ambient particles.

In general, we showed that the association between markers of exposure to traffic (time spent in traffic, number of trucks and automobiles and street density) and the FA was robust and not significantly affected by the different model specifications considered in our study. Time spent in traffic means more time exposed to increased concentration of smaller particles (tailpipe emission) that can easily reach the deeper portion of our respiratory tract, deposit and be retained. Other markers of traffic exposure, such as distance from home to a major road and local traffic density exhibited positive associations with carbon deposition but did not reach statistical significance. Residential concentration of particles is determined by indoor generation (e.g., cooking, vacuum cleaning, smoking) as well as by infiltration of outdoor particles (Diapouli et al., 2007; Morawska et al., 2013). Thus, residential activity patterns and home characteristics such as time spent at home, ventilation or type of stove used at home influence particulate matter levels and modify individual exposure. This highlights the importance of measuring daily activity and occupation for assessing individual exposure to particulate matter (Buonanno et al., 2014). Except for passive smoking and traffic density near home, we did not investigate exposure to indoor particles. Indeed, the levels of carbon deposited in the lungs of individuals who spent more time in traffic/commuting is higher than that resulting from passive smoking and, in some cases, reaches levels similar to those of current smokers. The characteristics of our study do not allow the 29

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Fig. 6. Comparative histological aspects in lungs from individuals with low and high exposure to ambient levels of air pollution in Sao Paulo. Top panels evidence histological aspects of pleural and subjacent pulmonary tissue in individuals with low (A) and high (B) exposure to air pollution. Note the deposition of black carbon in the pleural face (P). Arrows show the lymphatic vessels present in the interlobular connective tissue (IL) showing the existing anatomical connections between the pleural and intrapulmonary parenchyma, as well as the continuity of the draining systems of both compartments. Bottom panels indicate histopathological aspects of the region of the respiratory bronchiole (RB) in individuals with low (C) and high (D) exposure to ambient levels of air pollution. Note the presence of black carbon particles and marked distortion of normal centriacinar region in D.

Table 3 Effects of predictors variables in different stages of the fitting of the nonlinear regression models. Variables

Model 1 coef

Intercept Upper lobe Daily commuting (hours) Smoking amount Environmental tobacco smoke Age Years living in Sao Paulo Socioeconomic index Street density (3) Distance (4)

(1)

−1.582 0.310 0.048

Model 2 SE

(2)

0.057 0.034 0.015

(1)

p

coef

< 0.001 < 0.001 0.001

−1.752 0.319 0.044 0.005 0.110

Model 3 SE

(2)

0.069 0.034 0.014 0.001 0.082

(1)

p

coef

< 0.001 < 0.001 0.002 < 0.001 0.181

−3.149 0.317 0.046 0.005 0.105 0.019 0.000 −0.332

Model 4 SE

(2)

0.228 0.034 0.014 0.001 0.078 0.003 0.002 0.114

(1)

p

coef

< 0.001 < 0.001 < 0.001 < 0.001 0.177 < 0.001 0.922 0.004

−3.232 0.318 0.047 0.005 0.107 0.019 0.000 −0.395 7.364 0.000

Model 5 SE

(2)

0.255 0.034 0.013 0.001 0.077 0.003 0.002 0.123 5.871 0.000

(1)

SE

(2)

p

coef

p

< 0.001 < 0.001 0.001 < 0.001 0.165 < 0.001 0.890 0.001 0.210 0.414

−3.265 0.318 0.047 0.005

0.242 0.034 0.013 0.001

< 0.001 < 0.001 < 0.001 < 0.001

0.019

0.003

< 0.001

−0.368 9.170

0.114 5.527

0.001 0.097

(1): coefficient; (2): standard error; (3): street density in a buffer of 100 m around deceased's address; (4): distance from the residence to closest street of major traffic intensity. The coefficients of the explanatory variables are the logarithms of odds ratios; exponentiated coefficient is the amount by which FA/(1-FA) is multiplied with the increase of one unit in any predictor, maintaining the remaining ones at a fixed level. 30

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Appendix A. Supporting information

Our results may underestimate the dose to which individuals are exposed, since we measured only particles trapped in the surface of pulmonary tissue. Quantitative human tissue examination provided evidence that ambient particles can translocate from the lung into the circulation (Miller et al., 2017) and accumulate in organs such as the brain (Maher et al., 2016). In addition, we do not know whether the accumulation of particles in the lungs exhibits a saturation level, a factor that may limit the conclusions of our study in areas or conditions with higher levels of air pollution. An additional aspect to be considered is that we only measured particles trapped within the fibrotic foci elicited by particle-induced inflammation. Particles emitted by tobacco and traffic do have significant capacity to promote substantial remodeling of pulmonary structure, characterized by recruitment of inflammatory cells and fibrosis, mainly at centriacinar regions and areas of lympathic drainage such as peribronchiolar and perivascular interstitial space, interlobular septa and subpleural connective tissue. Although we conducted face-to-face interviews with the relatives of our subjects, the possibility of biases intrinsically associated with questionnaires and difficulties of reporting habits are of concern. However, we believe that such biases, although difficult to quantify, should not invalidate our results. In fact, the clear association between black carbon deposits and past smoking history (a kind of positive control) shows that, to some extent, recollection biases are not playing a major role. The design of the present study is not usual, and its potential merit resides on the unique conditions of Sao Paulo and its large autopsy service. To our knowledge, our study is one of the largest focusing on the accumulation of particles in the lung as the result of exposure to air pollution and the first to detect a significant association with time spent in traffic. Moreover, evaluation of the effect of commuters exposure to traffic has not been previously conducted in a large autopsy based study. Another implication of our findings is that the reduction of pollution-induced health burden is not entirely dependent on emission control but also depends on traffic fluidity. In conclusion, traffic exposure is a significant source of intake of air pollution and time spent in traffic is an efficient proxy variable to adjust for exposure variability in an urban setting. In addition, we postulate that policies aimed to reduce time spent in traffic (for instance, reducing traffic congestion) may be considered as an additional tool to reduce the dose of pollutants inhaled in megacities.

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Acknowledgments The authors thank the pathologists and technicians of the Post Mortem Verification Service of the University of Sao Paulo, the interviewers and the undergraduate students for their valuable assistance. Funding sources This work was supported by Sao Paulo Research Foundation (FAPESP), grants #13/21728-2; #16/23129-7, #16/22793-0, #16/ 03461-7 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant # 304126/2015-2. Ethics statement This study is part of the MetroHealth subproject of a project entitled The Use of Modern Autopsy Techniques to Investigate Human Diseases (MODAU) and was approved by the Research Ethics Committee of the University of Sao Paulo (number 537.195). Declarations of interest None. 31

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