Fresh tissues of brain, lung, olfactory bulb and olfactory epithelium were obtained from 30 individuals from the Post-Mortem Verification Service of São Paulo city (SVOC) (Table 1). In an SVO routine, a relative [next-of-kin (NOK)] claimed the body. A trained interviewer invited the NOK to participate in the study. Upon acceptance, the relative was invited to a private room where an informed consent form was signed authorizing the collection of tissue samples (Supplementary Material). Subsequently, a questionnaire was used to determine previous health conditions, residential address, sociodemographic details, life habits, smoking habits, occupation, time of residence in the Metropolitan Sao Paulo area (MSP), and time spent commuting. The inclusion criteria were: age equal or greater than 18 years, living in the MSP area for at least 3 months, having one close relative to provide reliable and complete information during the interview, and the absence of macroscopic alterations of the lungs, brain or the olfactory epithelium. This study was approved by the Research Ethics Committee of the University of Sao Paulo (number 2013/21728).

Sample preparation of human tissues

The tissue samples from brain (frontal lobe) and olfactory bulb, olfactory epithelium, lung (upper lobe) were placed separately in clean polyethylene bags and kept at −80 °C prior to transport to the Nuclear and Energy Research Institute (IPEN-CNEN/SP). Special care was taken during handling to avoid contamination using plastic tools and powder-free surgical gloves. The tissues were rinsed with purified water to remove the blood. Subsequently, the tissues were homogenized using a titanium knife and a hammer to grind cartilage of the olfactory epithelium. Samples were freeze-dried until their constant weight was obtained. In this process of freeze-drying mean percent weight losses were: 81.0 ± 7.1 for olfactory bulb, 71.9 ± 4.6 for olfactory epithelium, 82.0 ± 6.0 for lung and 79.7 ± 3.0 for brain. The dried samples were ground to a fine powder and placed in polyethylene vials, which were stored in a refrigerator.

Determination of 210Po in human samples

There are a limited number of reliable methods available for the determination of 210Po, the most commonly used being alpha particle spectrometry, due to its sensitivity and selectivity33. For the determination of 210Po in the tissue samples, approximately 0.5 g of each sample (dry weight) was used. Prior to the analysis, 100 μL of 209Po tracer of known activity (1.648 Bq/g) was added. For the acid dissolution of the sample, 10 mL of concentrated HNO 3 and hydrogen peroxide were added, under heating at 80 °C to avoid loss by volatilization of polonium isotopes. The solution was evaporated carefully to near dryness. This procedure was repeated until complete dissolution of the sample. The final solution was treated with concentrated HCl to eliminate nitrates. The final residue was dissolved in 0.5 M HCl and filtered with Millipore 0.1 μm membrane filters; 20% hydroxylamine hydrochloride was added to the solution. The pH was adjusted to 1.5 and the polonium was plated on a silver disc at 80 °C for 4 h, with agitation of the solution. The prepared sources were counted in a Canberra Alpha Analyst surface barrier detector for 150,000 s. For the determination of the detector alpha particle counting efficiency, a calibrated source of 241Am from Amersham, with activity of 5.55 kBq, was counted for 300 s. The counting efficiency of the detector varied from 22.54% to 26.96%.

Urban pollution exposures

We analyzed the association between 210Po and the following potential predictors of exposure to urban pollution: time living in Sao Paulo, daily commuting, and anthracosis index6. Anthracosis index was measured on the pleural surface of the lungs. 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. PhotoFigures of the pleura surface were taken with a high-resolution camera (Canon Power Shot SX400 IS). Using these images, the Fraction Area of Anthracosis (FA) was estimated by the point counting method34 with the aid of the software ImageJ (https://imagej.nih.gov/ij/docs/intro.html). For each of the four lobes, points falling on black patches (BP) and in the clean pleura (NBP) were counted separately and the fraction of anthracosis (FA) for each lung lobe determined applying the following formula:

$$FA=number\,of\,BP/(number\,of\,BP+number\,of\,NBP).$$

Other parameters

We also assessed the association between 210Po and smoking habits (smoker versus non-smoker), and socioeconomic index. We included socioeconomic index35 to identify vulnerable conditions related to health in the city of Sao Paulo. This index is based on 27 variables related to poverty, wealth, education, income, social and material deprivation, and cultural aspects35. This index ranges from −1 to 1, with higher values indicating better socioeconomic conditions. Details of these assessment methods were described elsewhere35.

Statistical analysis

All measurements of alpha particle activities from 210Po decay were expressed in Becquerel per kg of tissue (Bq/kg). Spearman’s correlation coefficient was used to evaluate the correlation between 210Po levels in the olfactory epithelium, olfactory bulb, frontal lobe, and lung tissues. Friedman’s test was applied to compare the distributions of 210Po in the different tissues. To find differences, pairwise comparisons were performed with Bonferroni correction. The Mann-Whitney test was applied to compare polonium distributions in male and female and in smokers and non-smokers. The association between the level of 210Po in the lung and anthracosis included adjustment for sex, age, smoking habits (smoker versus non-smoker), daily commuting (hr), years living in Sao Paulo, and socioeconomic index. Terms of interaction between sex and anthracosis and smoking and anthracosis were also added to the model. A residual analysis suggested the existence of one outlier. The analysis was repeated excluding this case. A robust regression model based on M-estimators was also considered. The model was fitted via the rlm function from the MASS library in the R software package. Due to the small sample size, the significance level was set at 0.10. The analysis was performed with Minitab (version 18) and R software package (R Development Core Team).

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 2013/21728).

Human experiment statement

All legal guardians signed the informed consent before the pathological procedures (Supplementary Material). This study was approved by the Research Ethics Committee of the University of Sao Paulo (number 2013/21728) in accordance with relevant regulations.