International Journal of Environmental Health Research, 2015 Vol. 25, No. 1, 67–80, http://dx.doi.org/10.1080/09603123.2014.893569
The suitability of EBC-Pb as a new biomarker to assess occupational exposure to lead Pedro M. Félixa*, Susana Marta Almeidaa, Cristiana Francoa, António Bugalho Almeidab, Carlos Lopesb, Maria Inês Clarob, Elsa Fragosob, Catarina Telesb, Hubert Th. Wolterbeekc and Teresa Pinheiroa a Campus Tecnológico e Nuclear, Dpt Física, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, Portugal; bDepartamento de Pneumologia, Hospital de Santa Maria Lisboa, Lisboa, Portugal; cDepartment of Radiation Science & Technology, Delft University of Technology, Delft, The Netherlands
(Received 3 May 2013; final version received 23 January 2014) Occupational exposure to lead (Pb) requires continuous surveillance to assure, as much as possible, safe and healthful working conditions. This study addresses the suitability of assessing Pb exposure in relevant workers using their exhaled breath condensate (EBC). This study enrolled workers of two different Pb processing industries characterized by moderate and high Pb exposure levels in the work environment, and a group of non-exposed individuals working in offices who served as baseline for Pb exposure. The EBC-Pb of workers reflected the Pb levels in the work environment of all three settings, although the relationship with B-Pb was not clear. The lack of correlation between EBC-Pb and B-Pb most probably indicates the time lag for Pb to enter in the two body pools. The EBC-Pb seems to reflect immediate exposure, providing a prompt signature of Pb in the environmental that may interact directly with the organ. By delivering short-term evaluation of exposure, EBC-Pb represents a clear advantage in biomonitoring and may become an interesting tool for estimating organ burden. Keywords: exhaled breath condensate; biomarker of exposure; lead; blood-Pb; occupational exposure
Introduction Present lead (Pb) exposures in working and outdoor environments are undoubtedly lower than several decades ago. However, occupational and environmental exposures to Pb continue to be a public health concern (Gurer-Orhan et al. 2004; Grover et al. 2010; Nie et al. 2011) due to its adverse effects on many organs’ functions. Exposure to this metal can cause haematological, gastrointestinal, rheumatologic, endocrine, neurological, renal and thyroid problems in humans (Pagliuca et al. 1990; Singh et al. 2000). Pb in the form of dust or fumes is mainly absorbed through respiratory and the digestive systems, reaching blood circulation, where it shows a high affinity to several erythrocytary proteins (Gonick 2011), and is distributed by several organs, although preferentially accumulated in calcified structures, such as bone and teeth (e.g. O’Flaherty 1993, 1995). Pb toxicity involves fundamental biochemical processes. These include the ability of Pb (i) to interfere in several enzymatic steps of haeme formation *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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by inhibiting δ-aminolevulinic acid dehydratase and ferrochelatase activity with effects in red blood cell survival and in a large number of metabolic pathways; (ii) to mimic the actions of calcium, which can affect osteogenic processes; (iii) to inhibit the conversion of vitamin D to 1,25-dihydroxyvitamin D3 with implications in calcium mobilization and bone development. Nowadays, the major Pb exposure hazards derive from exposure in industrial settings. Regulations impose strict rules, both to the industry and to the workers, to reduce the Pb levels in the work environment and the dose received by the worker. The workers’ exposure is usually assessed by measuring Pb in the blood (indirect biomonitoring). Early studies by Chamberlain et al. (1975) conducted in subjects exposed to Pb, either by intravenous administration or inhalation of fine particles, showed that 24 h after exposure the blood Pb concentration reached equilibrium (maximum stable value) in both tested conditions. Similar experimental designs also confirmed whole blood as a good mirror of Pb intake. However, in occupational settings where inhaled particles may vary considerably in size and composition (Park & Paik 2002; Pinheiro et al. 2011; Félix, Almeida et al. 2013), absorption routes may contribute unevenly to Pb concentrations in blood. Inhaled finer particles reach deeply into the respiratory system, facilitating the absorption, than coarser particles that deposit in the higher portion of the respiratory system (Bondesson et al. 2009). The coarser particles are more likely to enter the digestive system, through sputum secretion (that retains particles, being then removed by ciliated epithelia and reflections like sneezing or cough), than to be absorbed through the lungs (Geiser et al. 2005), although the lining fluids in the lung may enhance the agglomeration of fine particles facilitating their clearance through cough (Kendall et al. 2002). Thus, the proportion of particles of different size fractions that are inhaled and interact with the lung cannot be easily estimated. Subsequently, the primary inhaled particles or their constituents, which eventually reach circulation, follow different pathways at different times. These aspects prevent the true estimation of dose and limit biomonitoring through blood for long-term assessment in many conditions, such as the case of occupational exposure to Pb dust. Variations of Pb in blood of exposed individuals show a high time lag. The Pb in erythrocytes has a half-life of approximately 120 days, which is related to erythrocytes turn over (Hoet 2005). The Pb levels in blood may also be influenced by episodes of iron deficiency, low dietary calcium, bone reabsorption or osteoporosis, which may generate false positives (Heard & Chamberlain 1982; Marcus & Schwartz 1987). Several studies have explored the potential of exhaled breath condensate (EBC) as a tool for the monitoring of clinical conditions (Grob et al. 2008). Nevertheless, the use of EBC for assessing exposure to toxic metals in airborne particulate, fumes and other occupational emission products are limited. Some of these studies described the EBC quality to assess metal exposures by measuring changes in pH and condensed gases related to oxidative mechanisms (Caglieri et al. 2006; Hoffmeyer et al. 2012). A few others have quantitatively assessed direct exposure of workers to metals such as Cr (Goldoni et al. 2006), Co and W (Broding et al. 2009). The suitability of EBC to assess workers exposed to Pb dust has not been thoroughly examined. We have previously reported that Pb can be quantitatively measured in EBC of both non-exposed and occupationally exposed individuals (Pinheiro et al. 2011; Félix, Almeida et al. 2013; Félix, Franco et al. 2013), but the question of EBC delivering useful information about individual exposure to Pb has not been completely answered.
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Therefore, the aim of the present study is to examine (1) whether EBC-Pb reflects the exposure to Pb through time by repeatedly assessing workers during the working week and (2) the relationship between Pb in EBC and whole blood, which is the current bioindicator of Pb professional exposures. Materials and methods Industries and study groups This study enrolled workers from two industries that process lead: Industry 1 (Ind1) – battery recycling; and Industry 2 (Ind2) – battery manufacturing. The characterization of the industries and the work environment, and the methodology have been previously described (Félix, Almeida et al. 2013). Environmental and health regulations (transcript from European Union regulations, National Decree 274/89, 1989) are fully applied in both industries. Ind1 labours 24 h a day on three 8-h shifts and Ind2 operates only on two 8-h shifts per day. Workers from both factories labour successively during five days with two intercalary resting days between shifts. A non-exposed group of individuals working in offices not exposed to gases, dusts or fumes in their working activity were also enrolled. The working week consisted of 35 h, from Monday to Friday. This group provided a baseline for Pb biomonitoring in the exposed groups. The two groups of exposed individuals consisted of 15 effective workers of Ind1 and 77 of Ind2. These two groups of workers from Ind1 and Ind2 were exposed to different levels of Pb in the work environment for more than five years. The two industries and offices enabled the establishment of three levels of Pb exposure based on the mass concentration of particulate matter with aerodynamic diameter inferior to 10 μm (PM10), and the Pb concentration measured in this fraction, as described elsewhere (Félix, Almeida et al. 2013), is summarized in Table 1 as follows: (i) a high level of exposure in Ind1 (battery recycling), where the highest Pb levels were registered; (ii) a moderate level of Pb exposure in Ind2; (iii) a low level of Pb exposure in offices, which served as the baseline of the Pb occupational exposure. All the individuals recruited for this study gave their informed consent to participate in the study. Workers’ characterization The workers were also asked to fill a questionnaire reporting on demographics, smoking habits and clinical history concerning respiratory or other diseases. To assure the study would not be biased by health-related issues, the subjects were clinically evaluated by taking into account respiratory complaints, such as allergy, occasional cough, wheezing and sputum production, and their respiratory function was assessed by spirometry (Vitalograph Compact II spirometer, Ennis, Ireland). Two volumes, which is suitable for Table 1. Workplace characterization: PM10 concentration and Pb concentration in PM10 (Félix, Almeida et al. 2013).
PM10 (μg m−3) ± SE Pb–PM10 (μg m−3) ± SE
Offices (low level)
Industry 2 (intermediate level)
Industry 1 (high level)
25 ± 1 0.010 ± 0.001
125 ± 9 19 ± 4
1440 ± 193 518 ± 103
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carrying out measures to diagnose airway obstruction or restriction, can be obtained by exhaling into the spirometer, forced expiratory volume in the first second (FEV1) and forced vital capacity (FVC). The FEV1 and FVC are expressed as a percent of predicted values (for individuals of similar height, age, sex and weight). The ratio of these two volumes (FEV1/FVC), usually referred to as the Tiffeneau index, was calculated, as it is also useful in for diagnosis (Rabe et al. 2007). In addition, the exhaled nitric oxide (NO) was measured using a portable device, NIOX MINO (Aerocrine, Solna, Sweden). EBC sampling The EBC was collected using a commercial equipment (EcoScreen, Jager, Germany) as described elsewhere (Félix, Almeida et al. 2013). Briefly, the equipment had two unidirectional valves that prevented the mixing of inhaled and exhaled air in the collection tube and a functional saliva trap (Rosias et al. 2004; Broding et al. 2009). The temperature in the condenser was kept constant throughout the collection period, which guarantees the condensation of the droplets of exhaled air. Subjects were asked to breathe tidally for 15 min after rinsing their mouth with water. Sample collection was carried out at the factory’s occupational health unit. EBC of the non-exposed group was collected in a clean room (ISO7). Workers were evaluated in the beginning of the five-day working shift before starting work (a) and at the end of the last day of the shift (b). All EBC samples were pipetted into polypropylene containers, previously cleaned with HNO3 suprapur (20 % v/v) and acidified with 3 % of the same solution, prior to storage at −80 °C, according to the method described in Félix, Franco et al. (2013). Before sample acidification, a subsample was taken for pH measurement using a Hach H138 miniLab Elite pH metre. Determination of EBC-Pb The concentration of Pb in EBC was determined by an inductively coupled plasma mass spectrometry (ICP-MS) using an ELAN DRC-e equipment (PerkinElmer SCIEX, USA) operated at 1100 W (RF power) and with a nebulizer gas flow of 0.85 L min−1. The analyses were carried out with nickel cones and a Peltier-cooled quartz cyclonic spray chamber fitted for low-volume samples. For ICP-MS analysis, 500 μL of EBC samples was doped with 10 μg L−1 of Yttrium (Y) solution atomic absorption spectrometry (AAS Specpure® Y solution 1000 ± 10 μg mL−1 Alpha Aesar) and diluted at 1:5 v/v in ultrapure water (Milli-Q Element®) acidified at 1 % v/v with HNO3 suprapur (Merck) (Félix, Franco et al. 2013). A volume of 2.5 mL is the minimum required to perform an ICP-MS guaranteeing adequate conditions of sample analysis. Detailed calibration procedure and sample preparation were described elsewhere (Félix, Franco et al. 2013), and so was method validation and uncertainty estimation (Barreiros et al. 2013). Data were collected, processed and analysed with ELAN software v3.4. Determination of B-Pb Workers follow a screening scheme for blood Pb concentration according to National Regulations. The blood Pb concentration of the closest date to the EBC collection (20 days maximum difference) was used to minimize the time difference of Pb measurements in both blood and EBC matrices. The B-Pb concentrations were measured by AAS.
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Statistical analysis Exploratory analyses confirmed the absence of both variable normality (Shapiro Wilk) and homocedasticity (Levene’s test). Subsequently, Mann–Whitney U-tests were performed to determine the differences in EBC-Pb and B-Pb between industries and exposed vs. non-exposed, as well as to determine the influence of confounders for continuous variables (age, working years) and χ2 for categorical variables (smokers, gender). The Wilcoxon routine tested the differences between collection phases (a vs. b); for the latter, non-parametric tests and all descriptive and exploratory statists were run in Statistica (Statistica v9, Statsoft®, 2009). Relations of EBC-Pb with B-Pb were made with the collection phase b, corresponding to the post-exposure period (after the last day’s shift). All statistical tests were considered significant at p < 0.05. Results Demographic, clinical and physiological data of non-exposed and exposed workers are described in Table 2. The individuals enrolled in the present study worked and lived in the same geographical area and did not differ in age, smoking habits and number of working years. Workers of the Ind1 were only males, whereas in Ind2 22 % were women. In the non-exposed group, the percentage of both genders was similar.
Table 2. Demographic data on the workers of both industries and non-exposed group and clinical assessment of the workers of both industries and non-exposed group, according to smoking habits. Values include mean ± SD (when applicable). FEV1 – forced expiratory volume in the first second; FVC – forced vital capacity; Tiffeneau index = (FEV1/FVC); NO – exhaled nitric oxide.
Sex (m/f) Age (years) Working years Smokers (%) Allergic complaints (%) – All Non-smokers Smokers Respiratory symptoms (%) – All Non-smokers Smokers FVC (%) – All Non-smokers Smokers FEV1 (%) – All Non-smokers Smokers Tiffeneau index – All Non-smokers Smokers NO (ppb) – All Non-smokers Smokers
Non-Exposed (N = 52)
Industry 1 (N = 15)
Industry 2 (N = 77)
29/23 34 ± 8 – 24 – – – – – – – – – – – – – – – 20 ± 14 20 ± 14 20 ± 14
15/0 40 ± 8 13 ± 8 29 12 25 0 35 50 100 103 ± 16 106 ± 17 92 ± 1 103 ± 18 106 ± 19 92 ± 7 0.99 ± 0.06 0.99 ± 0.05 1.00 ± 0.09 18 ± 13 21 ± 15 9 ± 4#
64/17* 42 ± 10 14 ± 9 38 31 34 24 16* 16 17 89 ± 13* 88 ± 14 91 ± 11 90 ± 14* 90 ± 14 89 ± 13 1.0 ± 0.1 1.03 ± 0.09 0.98 ± 0.09 15 ± 11 18 ± 13 11 ± 4#
*Significant differences between both industries (Mann–Whitney results); # Significant differences to non-smokers (χ2 results).
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The clinical evaluation of exposed workers did not reveal any major clinical complications. Exposed workers reported respiratory symptoms, which were more prevalent in Ind1. The respiratory function data did not reflect any important physiological alterations, namely FEV1 and FVC (expressed in percentage of predicted), and the Tiffeneau index (Table 2). All spirometry parameters were considered within the expected normal values according to the standard criteria set by the Global Initiative for Obstructive Lung Disease (World Health Organization). The values of FEV1 and FVC were consistently lower in Ind2 when compared to Ind1. However, the statistically significant decrease of FEV1 observed in Ind2 relative to Ind1 workers lacked clinical relevance. The clinical and physiological evaluation of exposed workers had taken into account the possible aggravation of health conditions caused by smoking habits. The influence of smoking in respiratory function values and clinical evaluation was assessed. Individuals smoking for at least one year were considered smokers. The prevalence of smoking in both groups did not produce significant alterations in the respiratory function as can be inferred from the data listed in Table 2. The workers’ health examination comprised also NO measurements in exhaled air. The groups did not differ in terms of NO concentration. However, significant differences between smokers and non-smokers were found. The NO concentration in the exhaled breath of Pb exposed workers (Ind1 and Ind2) who were smokers was significantly lower than in non-smokers, as reported in Table 2. Opposite, no differences were observed between smokers and non-smokers for the group of non-exposed individuals working in offices (Table 2). Table 3 lists Pb concentrations in EBC and whole blood of the studied groups. The EBC-Pb of the three groups reflected the different levels of exposure: EBC-Pb was highest in workers of Ind1 and lowest in non-exposed. An increment of the Pb concentration in EBC of Ind1 and Ind2 workers was observed at the end of the working week. This increment was only significant in Ind2. B-Pb also showed a higher mean value in Ind1, which significantly differed for Ind2. Either for EBC-Pb or B-Pb, the differences between the groups and along the working week were independent of age, working years or smoking habits, the latter being illustrated in Figure 1. A minor increase of B-Pb with increasing age was observed, although the trend was not statistically significant (ρ = 0.19, p = 0.079). For each EBC sample, the pH was recorded immediately after collection as referred previously. No differences were observed in pH values in the three groups. However, when differences of pH were assessed for smoking habits (Figure 2), Ind1 showed a Table 3. Average lead (Pb) concentrations in EBC (μg L−1 ± SE) in workers from Industry 1 (N = 15), Industry 2 (N = 77) and non-exposed group (N = 52); and Blood (μg dL−1 ± SE) in workers from Industry 1 and Industry 2. For EBC-Pb: (a) before starting the shift, first day of the week; (b) end of the shift, last day of the week. EBC-Pb a X (μg L−1) ± SE Non-Exposed Industry 1 Industry 2 *
28 ± 5*¥ 1.6 ± 0.2*¥§
0.97 ± 0.25
EBC-Pb b X (μg L−1) ± SE
X (μg dL−1) ± SE
36 ± 7*¥ 2.3 ± 0.3*¥§
– 43 ± 4¥ 19 ± 0.8¥
Significant differences to non-exposed ( p < 0.001); Significant differences between industries ( p < 0.001); § Significant differences between collection a and b ( p < 0.001). ¥
B-Pb
Figure 1. Whisker plot depicting mean post-exposure values (±SE) of EBC-Pb for smokers and non-smokers of workers from (a) Ind1, (b) Ind2 and (c) nonexposed, with no significant differences.
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Figure 2. Whisker plot depicting mean post-exposure values (±SE) of pH for smokers and non-smokers of workers from (a) Ind1, (b) Ind2 and (c) nonexposed, with significant differences in (a) Ind1 (p < 0.05).
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Figure 3. Scatter plot depicting (a) Industry 1 and (b) industry 2 values of B-Pb (μg dL−1) against EBC-Pb (μg L−1) and respective trend lines.
significant decrease of pH in smokers when compared to non-smokers (smokers: 6.51 ± 1.13; non-smokers: 7.55 ± 0.67; p < 0.05). However, the EBC pH did not influence the Pb concentration in EBC. No significant correlations were found between B-Pb and EBC-Pb for both Ind1 and Ind2 (Figure 3). For Ind1, the correlation between EBC-Pb and B-Pb was ρ = 0.382, p = 0.08, whereas in Ind2 the correlation value was ρ = 0.124, p = 0.14. Although significance was not reached, Ind1 showed a closer proximity to a linear correlation than Ind2. It is worth noting that the maximum legal value of B-Pb is 70 μg dL−1, (compulsory withdrawal from work) and none of the workers presented such levels. Discussion The present work demonstrated the ability of EBC to assess professional exposure to Pb. The concentrations of Pb measured in EBC reflected the exposure levels of Pb in the workplace. As the concentration of Pb associated with PM increased, the levels of Pb in EBC also increased. Although smoking habits were related to documentation of respiratory symptoms and significantly influenced respiratory function indicators, such as NO concentration in exhaled breath, no influence on Pb concentration in EBC could be found. Also, the Pb concentration in EBC was not influenced by age, gender and working years. This suggests that during ventilation the settlement of inhaled particles, and especially those containing Pb, is independent of individual characteristics and risk factors, such as smoking. The increment of Pb concentration in the EBC of workers during the working week period of time was statistically relevant in Ind2, whereas in Ind1, where Pb exposure was high, the concentrations of Pb in EBC did not significantly vary. The relative changes in EBC-Pb from beginning to end of the shift were similar in both industries (ratio of 1.3 ± 0.3 in Ind1 and 1.4 ± 0.3 in Ind2, as can be inferred from Table 3), suggesting that Pb lung clearance during the two-day resting period after a week of exposure was similar. However, in Ind1, the statistical power to resolve the small differences in Pb levels between the two evaluation time points was not sufficient. This suggests that regular exposure to high levels of Pb favoured a high residence time of Pb in the airways.
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It was also observed that Pb concentration increases in EBC and in blood according to the exposure level of Pb in the work environment. However, the variations in Pb concentrations in EBC and blood were not correlated. This can be an acceptable result, considering that the retention time in the two body compartments differ. The Pb may be retained in red blood cells for their entire life cycle, approximately 120 days (e.g. Rabinowitz et al. 1976; Chamberlain et al. 1978; Moreira & Moreira 2004), whereas retention in the lung may be ruled by different factors. The Pb concentration in EBC contains the signature of the external environment, which, in principle, is not limited. The retention of particles in the lungs is primarily based on mechanical interactions (e.g. impact of particles in lung lining fluid) rather than biochemical interactions with specific kinetics occurring in blood. Thus, measurements in EBC may give the internal dose level practically in real time and this may justify, per se, the lack or relatively weak correlation between EBC and blood. Therefore, EBC may be a fast-term indicator of exposure and blood a medium-term indicator, with several contributions accounting for the time lag between exposure and the Pb found in blood. Ind1 data for B-Pb and EBC-Pb were statistically closer to a linear correlation than Ind2 data. The proportion of fine particles (PM2.5) emitted by Ind1 was, in fact, considerably higher than that found for Ind2 (Félix, Almeida et al. 2013). Particle size does not hamper the EBC-Pb analysis, which translates exposure of different occupational settings (Félix, Almeida et al. 2013), although it may influence the assessment through B-Pb. The fact that finer particles may be easily absorbed into the blood through lungs (Snee et al. 1985; ATSDR 1999) offers a faster response of B-Pb to exposure and, thus, closer proximity to a linear correlation with EBC-Pb. Earlier studies reported on the rapid uptake of inhaled fine particles seem to corroborate this hypothesis (Chamberlain et al. 1975), suggesting that the absorption of Pb, present in the inhaled particulate matter, from the lungs into the blood circulation can be modulated by the relative abundance of coarse and fine particles in the environment (IPCS 1995). As the Pb concentration increased in the work environment, the increment of EBC-Pb was higher than in B-Pb. This seems to indicate that the retention of Pb in blood may slow down or decrease during exposure to high levels of Pb in PM, suggesting that B-Pb would be a biomarker more suitable for moderate and low levels of exposure. In these extreme conditions, the Pb concentration in blood may be influenced by limiting factors, such as, absorption and transport rates, and saturation of Pb-binding sites in erythrocytes and carrier proteins in serum (Fleming et al. 1997; Skerfving & Bergdahl 2007). Thus, the simultaneous determination of Pb in blood cells, serum and plasma fractions would help elucidate the absorption. The levels of interference in EBC-Pb and B-Pb are schematized in Figure 4. Exchanges between blood, organs and tissues are expectable. However blood–lung transitions are unlikely. Thus, EBC is not influenced by confounders and this is an advantage over blood in terms of a biomarker. EBC is apparently a matrix with less probability for bias than whole blood. In fact, a peak in B-Pb does not necessarily mean a strong and recent deposit in the lungs. On the other hand, a low B-Pb does not mean that the subject is not highly exposed at the moment (OSHA 1993; ATSDR 2007). Moreover, with the advancing age of exposed workers, and reduction in bone mass, the levels of B-Pb are potentially and increasingly biased by the release of Pb accumulated in bone (Silbergeld et al. 1988; O’Flaherty 1993; Gulson et al. 1996). The levels of B-Pb have a higher probability of being influenced by endogenous Pb. Presently, both EBC-Pb and B-Pb cannot indicate the dose of intake. However, by using EBC as a proxy to assess the particles settled in the airways, there is a high
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Figure 4. Schematic representation of the Pb mobilization pathways based on O’Flaherty (1993, 1995), including main routes of excretion (black arrows). Strikes over the arrow indicate an inexistent or negligible pathway.
potential to obtain dose information. Further studies should explore the capability of EBC to express the dose of intake by inhalation for an exposure period of interest. This would involve a follow-up of exposed individuals to different levels of Pb through inhalation, with periodic and simultaneous measurements of EBC-Pb and airborne Pb. Measurement of biomarkers of inflammation, including leukotriene B4 (Montuschi 2009) and metabolites (Montuschi et al. 2012; Motta et al. 2012), or 15-F2t-isoprostane, as a marker of oxidative stress, in EBC (Lucidi et al. 2008; Barreto et al. 2009; Montuschi et al. 2010) might be useful for assessing Pb pulmonary toxicity. Detecting breath VOC profiles by e-noses might also be a novel approach for identifying lung inflammatory changes (Fens et al. 2011; Bofan et al. 2013; Montuschi et al. 2013). Such data would be of utmost importance to model B-Pb changes and to quantify the contribution of sources of intake in blood. EBC proved to be useful for occupational exposure assessment and further trial research programmes would help establishing EBC as a complementary test in workers’ biomonitoring. The B-Pb is useful as a complementary tool to assess toxicity, given its ability to reflect systemic Pb, as in episodic endogenous Pb releases, which are factual risks, particularly on retired workers (Erkkilä et al. 1992; Gerhardsson et al. 1993). On the other hand, EBC-Pb is useful to assess Pb intake by inhalation and to estimate the burden of the target organ in continuously exposed subjects. The potential of EBC may offer a valid contribution to intake quantification, considering the amount of time the worker has been exposed to Pb. Hence, EBC may help predicting temporal limits of exposure and preventing long-term hazardous health outcomes, relatively common in chronic Pb exposure. Conclusions Ideally, the identification of exposure is done through a biomarker that reduces probable confounders and has the most reduced number of sources. The potential of EBC-Pb as a
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biomarker of exposure to Pb in occupational settings was emphasized in this study by (i) the ability of EBC to assess different levels of exposure; (ii) the minor influence of confounders, such as gender, age, smoking habits and working years; (iii) non-invasive characteristics, which allow a continuous monitoring of a subject by serial sampling; (iv) and analytically undemanding processes, resulting in expedite analysis and little sample manipulation. These features are important to validate EBC as a biomarker. Moreover, EBC opens the possibility to determine the dose by measuring the accumulation through time of a relevant exposure indicator and to assess variable exposures, such as work-post change. This makes EBC an attractive matrix in occupational exposure studies. Acknowledgements The authors gratefully acknowledge Fundação para a Ciência e Tecnologia (FCT) for funding the project PTDC/AMB/65828/2006 – Exhaled breath condensate: a tool for non-invasive evaluation of pollutant exposure? The authors thank Dr Marta Santos for the fruitful discussions and expertise in the ICP-MS.
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The suitability of EBC-Pb as a new biomarker to assess occupational exposure to lead Pedro M. Félixa*, Susana Marta Almeidaa, Cristiana Francoa, António Bugalho Almeidab, Carlos Lopesb, Maria Inês Clarob, Elsa Fragosob, Catarina Telesb, Hubert Th. Wolterbeekc and Teresa Pinheiroa a Campus Tecnológico e Nuclear, Dpt Física, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, Portugal; bDepartamento de Pneumologia, Hospital de Santa Maria Lisboa, Lisboa, Portugal; cDepartment of Radiation Science & Technology, Delft University of Technology, Delft, The Netherlands
(Received 3 May 2013; final version received 23 January 2014) Occupational exposure to lead (Pb) requires continuous surveillance to assure, as much as possible, safe and healthful working conditions. This study addresses the suitability of assessing Pb exposure in relevant workers using their exhaled breath condensate (EBC). This study enrolled workers of two different Pb processing industries characterized by moderate and high Pb exposure levels in the work environment, and a group of non-exposed individuals working in offices who served as baseline for Pb exposure. The EBC-Pb of workers reflected the Pb levels in the work environment of all three settings, although the relationship with B-Pb was not clear. The lack of correlation between EBC-Pb and B-Pb most probably indicates the time lag for Pb to enter in the two body pools. The EBC-Pb seems to reflect immediate exposure, providing a prompt signature of Pb in the environmental that may interact directly with the organ. By delivering short-term evaluation of exposure, EBC-Pb represents a clear advantage in biomonitoring and may become an interesting tool for estimating organ burden. Keywords: exhaled breath condensate; biomarker of exposure; lead; blood-Pb; occupational exposure
Introduction Present lead (Pb) exposures in working and outdoor environments are undoubtedly lower than several decades ago. However, occupational and environmental exposures to Pb continue to be a public health concern (Gurer-Orhan et al. 2004; Grover et al. 2010; Nie et al. 2011) due to its adverse effects on many organs’ functions. Exposure to this metal can cause haematological, gastrointestinal, rheumatologic, endocrine, neurological, renal and thyroid problems in humans (Pagliuca et al. 1990; Singh et al. 2000). Pb in the form of dust or fumes is mainly absorbed through respiratory and the digestive systems, reaching blood circulation, where it shows a high affinity to several erythrocytary proteins (Gonick 2011), and is distributed by several organs, although preferentially accumulated in calcified structures, such as bone and teeth (e.g. O’Flaherty 1993, 1995). Pb toxicity involves fundamental biochemical processes. These include the ability of Pb (i) to interfere in several enzymatic steps of haeme formation *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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by inhibiting δ-aminolevulinic acid dehydratase and ferrochelatase activity with effects in red blood cell survival and in a large number of metabolic pathways; (ii) to mimic the actions of calcium, which can affect osteogenic processes; (iii) to inhibit the conversion of vitamin D to 1,25-dihydroxyvitamin D3 with implications in calcium mobilization and bone development. Nowadays, the major Pb exposure hazards derive from exposure in industrial settings. Regulations impose strict rules, both to the industry and to the workers, to reduce the Pb levels in the work environment and the dose received by the worker. The workers’ exposure is usually assessed by measuring Pb in the blood (indirect biomonitoring). Early studies by Chamberlain et al. (1975) conducted in subjects exposed to Pb, either by intravenous administration or inhalation of fine particles, showed that 24 h after exposure the blood Pb concentration reached equilibrium (maximum stable value) in both tested conditions. Similar experimental designs also confirmed whole blood as a good mirror of Pb intake. However, in occupational settings where inhaled particles may vary considerably in size and composition (Park & Paik 2002; Pinheiro et al. 2011; Félix, Almeida et al. 2013), absorption routes may contribute unevenly to Pb concentrations in blood. Inhaled finer particles reach deeply into the respiratory system, facilitating the absorption, than coarser particles that deposit in the higher portion of the respiratory system (Bondesson et al. 2009). The coarser particles are more likely to enter the digestive system, through sputum secretion (that retains particles, being then removed by ciliated epithelia and reflections like sneezing or cough), than to be absorbed through the lungs (Geiser et al. 2005), although the lining fluids in the lung may enhance the agglomeration of fine particles facilitating their clearance through cough (Kendall et al. 2002). Thus, the proportion of particles of different size fractions that are inhaled and interact with the lung cannot be easily estimated. Subsequently, the primary inhaled particles or their constituents, which eventually reach circulation, follow different pathways at different times. These aspects prevent the true estimation of dose and limit biomonitoring through blood for long-term assessment in many conditions, such as the case of occupational exposure to Pb dust. Variations of Pb in blood of exposed individuals show a high time lag. The Pb in erythrocytes has a half-life of approximately 120 days, which is related to erythrocytes turn over (Hoet 2005). The Pb levels in blood may also be influenced by episodes of iron deficiency, low dietary calcium, bone reabsorption or osteoporosis, which may generate false positives (Heard & Chamberlain 1982; Marcus & Schwartz 1987). Several studies have explored the potential of exhaled breath condensate (EBC) as a tool for the monitoring of clinical conditions (Grob et al. 2008). Nevertheless, the use of EBC for assessing exposure to toxic metals in airborne particulate, fumes and other occupational emission products are limited. Some of these studies described the EBC quality to assess metal exposures by measuring changes in pH and condensed gases related to oxidative mechanisms (Caglieri et al. 2006; Hoffmeyer et al. 2012). A few others have quantitatively assessed direct exposure of workers to metals such as Cr (Goldoni et al. 2006), Co and W (Broding et al. 2009). The suitability of EBC to assess workers exposed to Pb dust has not been thoroughly examined. We have previously reported that Pb can be quantitatively measured in EBC of both non-exposed and occupationally exposed individuals (Pinheiro et al. 2011; Félix, Almeida et al. 2013; Félix, Franco et al. 2013), but the question of EBC delivering useful information about individual exposure to Pb has not been completely answered.
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Therefore, the aim of the present study is to examine (1) whether EBC-Pb reflects the exposure to Pb through time by repeatedly assessing workers during the working week and (2) the relationship between Pb in EBC and whole blood, which is the current bioindicator of Pb professional exposures. Materials and methods Industries and study groups This study enrolled workers from two industries that process lead: Industry 1 (Ind1) – battery recycling; and Industry 2 (Ind2) – battery manufacturing. The characterization of the industries and the work environment, and the methodology have been previously described (Félix, Almeida et al. 2013). Environmental and health regulations (transcript from European Union regulations, National Decree 274/89, 1989) are fully applied in both industries. Ind1 labours 24 h a day on three 8-h shifts and Ind2 operates only on two 8-h shifts per day. Workers from both factories labour successively during five days with two intercalary resting days between shifts. A non-exposed group of individuals working in offices not exposed to gases, dusts or fumes in their working activity were also enrolled. The working week consisted of 35 h, from Monday to Friday. This group provided a baseline for Pb biomonitoring in the exposed groups. The two groups of exposed individuals consisted of 15 effective workers of Ind1 and 77 of Ind2. These two groups of workers from Ind1 and Ind2 were exposed to different levels of Pb in the work environment for more than five years. The two industries and offices enabled the establishment of three levels of Pb exposure based on the mass concentration of particulate matter with aerodynamic diameter inferior to 10 μm (PM10), and the Pb concentration measured in this fraction, as described elsewhere (Félix, Almeida et al. 2013), is summarized in Table 1 as follows: (i) a high level of exposure in Ind1 (battery recycling), where the highest Pb levels were registered; (ii) a moderate level of Pb exposure in Ind2; (iii) a low level of Pb exposure in offices, which served as the baseline of the Pb occupational exposure. All the individuals recruited for this study gave their informed consent to participate in the study. Workers’ characterization The workers were also asked to fill a questionnaire reporting on demographics, smoking habits and clinical history concerning respiratory or other diseases. To assure the study would not be biased by health-related issues, the subjects were clinically evaluated by taking into account respiratory complaints, such as allergy, occasional cough, wheezing and sputum production, and their respiratory function was assessed by spirometry (Vitalograph Compact II spirometer, Ennis, Ireland). Two volumes, which is suitable for Table 1. Workplace characterization: PM10 concentration and Pb concentration in PM10 (Félix, Almeida et al. 2013).
PM10 (μg m−3) ± SE Pb–PM10 (μg m−3) ± SE
Offices (low level)
Industry 2 (intermediate level)
Industry 1 (high level)
25 ± 1 0.010 ± 0.001
125 ± 9 19 ± 4
1440 ± 193 518 ± 103
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carrying out measures to diagnose airway obstruction or restriction, can be obtained by exhaling into the spirometer, forced expiratory volume in the first second (FEV1) and forced vital capacity (FVC). The FEV1 and FVC are expressed as a percent of predicted values (for individuals of similar height, age, sex and weight). The ratio of these two volumes (FEV1/FVC), usually referred to as the Tiffeneau index, was calculated, as it is also useful in for diagnosis (Rabe et al. 2007). In addition, the exhaled nitric oxide (NO) was measured using a portable device, NIOX MINO (Aerocrine, Solna, Sweden). EBC sampling The EBC was collected using a commercial equipment (EcoScreen, Jager, Germany) as described elsewhere (Félix, Almeida et al. 2013). Briefly, the equipment had two unidirectional valves that prevented the mixing of inhaled and exhaled air in the collection tube and a functional saliva trap (Rosias et al. 2004; Broding et al. 2009). The temperature in the condenser was kept constant throughout the collection period, which guarantees the condensation of the droplets of exhaled air. Subjects were asked to breathe tidally for 15 min after rinsing their mouth with water. Sample collection was carried out at the factory’s occupational health unit. EBC of the non-exposed group was collected in a clean room (ISO7). Workers were evaluated in the beginning of the five-day working shift before starting work (a) and at the end of the last day of the shift (b). All EBC samples were pipetted into polypropylene containers, previously cleaned with HNO3 suprapur (20 % v/v) and acidified with 3 % of the same solution, prior to storage at −80 °C, according to the method described in Félix, Franco et al. (2013). Before sample acidification, a subsample was taken for pH measurement using a Hach H138 miniLab Elite pH metre. Determination of EBC-Pb The concentration of Pb in EBC was determined by an inductively coupled plasma mass spectrometry (ICP-MS) using an ELAN DRC-e equipment (PerkinElmer SCIEX, USA) operated at 1100 W (RF power) and with a nebulizer gas flow of 0.85 L min−1. The analyses were carried out with nickel cones and a Peltier-cooled quartz cyclonic spray chamber fitted for low-volume samples. For ICP-MS analysis, 500 μL of EBC samples was doped with 10 μg L−1 of Yttrium (Y) solution atomic absorption spectrometry (AAS Specpure® Y solution 1000 ± 10 μg mL−1 Alpha Aesar) and diluted at 1:5 v/v in ultrapure water (Milli-Q Element®) acidified at 1 % v/v with HNO3 suprapur (Merck) (Félix, Franco et al. 2013). A volume of 2.5 mL is the minimum required to perform an ICP-MS guaranteeing adequate conditions of sample analysis. Detailed calibration procedure and sample preparation were described elsewhere (Félix, Franco et al. 2013), and so was method validation and uncertainty estimation (Barreiros et al. 2013). Data were collected, processed and analysed with ELAN software v3.4. Determination of B-Pb Workers follow a screening scheme for blood Pb concentration according to National Regulations. The blood Pb concentration of the closest date to the EBC collection (20 days maximum difference) was used to minimize the time difference of Pb measurements in both blood and EBC matrices. The B-Pb concentrations were measured by AAS.
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Statistical analysis Exploratory analyses confirmed the absence of both variable normality (Shapiro Wilk) and homocedasticity (Levene’s test). Subsequently, Mann–Whitney U-tests were performed to determine the differences in EBC-Pb and B-Pb between industries and exposed vs. non-exposed, as well as to determine the influence of confounders for continuous variables (age, working years) and χ2 for categorical variables (smokers, gender). The Wilcoxon routine tested the differences between collection phases (a vs. b); for the latter, non-parametric tests and all descriptive and exploratory statists were run in Statistica (Statistica v9, Statsoft®, 2009). Relations of EBC-Pb with B-Pb were made with the collection phase b, corresponding to the post-exposure period (after the last day’s shift). All statistical tests were considered significant at p < 0.05. Results Demographic, clinical and physiological data of non-exposed and exposed workers are described in Table 2. The individuals enrolled in the present study worked and lived in the same geographical area and did not differ in age, smoking habits and number of working years. Workers of the Ind1 were only males, whereas in Ind2 22 % were women. In the non-exposed group, the percentage of both genders was similar.
Table 2. Demographic data on the workers of both industries and non-exposed group and clinical assessment of the workers of both industries and non-exposed group, according to smoking habits. Values include mean ± SD (when applicable). FEV1 – forced expiratory volume in the first second; FVC – forced vital capacity; Tiffeneau index = (FEV1/FVC); NO – exhaled nitric oxide.
Sex (m/f) Age (years) Working years Smokers (%) Allergic complaints (%) – All Non-smokers Smokers Respiratory symptoms (%) – All Non-smokers Smokers FVC (%) – All Non-smokers Smokers FEV1 (%) – All Non-smokers Smokers Tiffeneau index – All Non-smokers Smokers NO (ppb) – All Non-smokers Smokers
Non-Exposed (N = 52)
Industry 1 (N = 15)
Industry 2 (N = 77)
29/23 34 ± 8 – 24 – – – – – – – – – – – – – – – 20 ± 14 20 ± 14 20 ± 14
15/0 40 ± 8 13 ± 8 29 12 25 0 35 50 100 103 ± 16 106 ± 17 92 ± 1 103 ± 18 106 ± 19 92 ± 7 0.99 ± 0.06 0.99 ± 0.05 1.00 ± 0.09 18 ± 13 21 ± 15 9 ± 4#
64/17* 42 ± 10 14 ± 9 38 31 34 24 16* 16 17 89 ± 13* 88 ± 14 91 ± 11 90 ± 14* 90 ± 14 89 ± 13 1.0 ± 0.1 1.03 ± 0.09 0.98 ± 0.09 15 ± 11 18 ± 13 11 ± 4#
*Significant differences between both industries (Mann–Whitney results); # Significant differences to non-smokers (χ2 results).
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The clinical evaluation of exposed workers did not reveal any major clinical complications. Exposed workers reported respiratory symptoms, which were more prevalent in Ind1. The respiratory function data did not reflect any important physiological alterations, namely FEV1 and FVC (expressed in percentage of predicted), and the Tiffeneau index (Table 2). All spirometry parameters were considered within the expected normal values according to the standard criteria set by the Global Initiative for Obstructive Lung Disease (World Health Organization). The values of FEV1 and FVC were consistently lower in Ind2 when compared to Ind1. However, the statistically significant decrease of FEV1 observed in Ind2 relative to Ind1 workers lacked clinical relevance. The clinical and physiological evaluation of exposed workers had taken into account the possible aggravation of health conditions caused by smoking habits. The influence of smoking in respiratory function values and clinical evaluation was assessed. Individuals smoking for at least one year were considered smokers. The prevalence of smoking in both groups did not produce significant alterations in the respiratory function as can be inferred from the data listed in Table 2. The workers’ health examination comprised also NO measurements in exhaled air. The groups did not differ in terms of NO concentration. However, significant differences between smokers and non-smokers were found. The NO concentration in the exhaled breath of Pb exposed workers (Ind1 and Ind2) who were smokers was significantly lower than in non-smokers, as reported in Table 2. Opposite, no differences were observed between smokers and non-smokers for the group of non-exposed individuals working in offices (Table 2). Table 3 lists Pb concentrations in EBC and whole blood of the studied groups. The EBC-Pb of the three groups reflected the different levels of exposure: EBC-Pb was highest in workers of Ind1 and lowest in non-exposed. An increment of the Pb concentration in EBC of Ind1 and Ind2 workers was observed at the end of the working week. This increment was only significant in Ind2. B-Pb also showed a higher mean value in Ind1, which significantly differed for Ind2. Either for EBC-Pb or B-Pb, the differences between the groups and along the working week were independent of age, working years or smoking habits, the latter being illustrated in Figure 1. A minor increase of B-Pb with increasing age was observed, although the trend was not statistically significant (ρ = 0.19, p = 0.079). For each EBC sample, the pH was recorded immediately after collection as referred previously. No differences were observed in pH values in the three groups. However, when differences of pH were assessed for smoking habits (Figure 2), Ind1 showed a Table 3. Average lead (Pb) concentrations in EBC (μg L−1 ± SE) in workers from Industry 1 (N = 15), Industry 2 (N = 77) and non-exposed group (N = 52); and Blood (μg dL−1 ± SE) in workers from Industry 1 and Industry 2. For EBC-Pb: (a) before starting the shift, first day of the week; (b) end of the shift, last day of the week. EBC-Pb a X (μg L−1) ± SE Non-Exposed Industry 1 Industry 2 *
28 ± 5*¥ 1.6 ± 0.2*¥§
0.97 ± 0.25
EBC-Pb b X (μg L−1) ± SE
X (μg dL−1) ± SE
36 ± 7*¥ 2.3 ± 0.3*¥§
– 43 ± 4¥ 19 ± 0.8¥
Significant differences to non-exposed ( p < 0.001); Significant differences between industries ( p < 0.001); § Significant differences between collection a and b ( p < 0.001). ¥
B-Pb
Figure 1. Whisker plot depicting mean post-exposure values (±SE) of EBC-Pb for smokers and non-smokers of workers from (a) Ind1, (b) Ind2 and (c) nonexposed, with no significant differences.
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Figure 2. Whisker plot depicting mean post-exposure values (±SE) of pH for smokers and non-smokers of workers from (a) Ind1, (b) Ind2 and (c) nonexposed, with significant differences in (a) Ind1 (p < 0.05).
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Figure 3. Scatter plot depicting (a) Industry 1 and (b) industry 2 values of B-Pb (μg dL−1) against EBC-Pb (μg L−1) and respective trend lines.
significant decrease of pH in smokers when compared to non-smokers (smokers: 6.51 ± 1.13; non-smokers: 7.55 ± 0.67; p < 0.05). However, the EBC pH did not influence the Pb concentration in EBC. No significant correlations were found between B-Pb and EBC-Pb for both Ind1 and Ind2 (Figure 3). For Ind1, the correlation between EBC-Pb and B-Pb was ρ = 0.382, p = 0.08, whereas in Ind2 the correlation value was ρ = 0.124, p = 0.14. Although significance was not reached, Ind1 showed a closer proximity to a linear correlation than Ind2. It is worth noting that the maximum legal value of B-Pb is 70 μg dL−1, (compulsory withdrawal from work) and none of the workers presented such levels. Discussion The present work demonstrated the ability of EBC to assess professional exposure to Pb. The concentrations of Pb measured in EBC reflected the exposure levels of Pb in the workplace. As the concentration of Pb associated with PM increased, the levels of Pb in EBC also increased. Although smoking habits were related to documentation of respiratory symptoms and significantly influenced respiratory function indicators, such as NO concentration in exhaled breath, no influence on Pb concentration in EBC could be found. Also, the Pb concentration in EBC was not influenced by age, gender and working years. This suggests that during ventilation the settlement of inhaled particles, and especially those containing Pb, is independent of individual characteristics and risk factors, such as smoking. The increment of Pb concentration in the EBC of workers during the working week period of time was statistically relevant in Ind2, whereas in Ind1, where Pb exposure was high, the concentrations of Pb in EBC did not significantly vary. The relative changes in EBC-Pb from beginning to end of the shift were similar in both industries (ratio of 1.3 ± 0.3 in Ind1 and 1.4 ± 0.3 in Ind2, as can be inferred from Table 3), suggesting that Pb lung clearance during the two-day resting period after a week of exposure was similar. However, in Ind1, the statistical power to resolve the small differences in Pb levels between the two evaluation time points was not sufficient. This suggests that regular exposure to high levels of Pb favoured a high residence time of Pb in the airways.
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It was also observed that Pb concentration increases in EBC and in blood according to the exposure level of Pb in the work environment. However, the variations in Pb concentrations in EBC and blood were not correlated. This can be an acceptable result, considering that the retention time in the two body compartments differ. The Pb may be retained in red blood cells for their entire life cycle, approximately 120 days (e.g. Rabinowitz et al. 1976; Chamberlain et al. 1978; Moreira & Moreira 2004), whereas retention in the lung may be ruled by different factors. The Pb concentration in EBC contains the signature of the external environment, which, in principle, is not limited. The retention of particles in the lungs is primarily based on mechanical interactions (e.g. impact of particles in lung lining fluid) rather than biochemical interactions with specific kinetics occurring in blood. Thus, measurements in EBC may give the internal dose level practically in real time and this may justify, per se, the lack or relatively weak correlation between EBC and blood. Therefore, EBC may be a fast-term indicator of exposure and blood a medium-term indicator, with several contributions accounting for the time lag between exposure and the Pb found in blood. Ind1 data for B-Pb and EBC-Pb were statistically closer to a linear correlation than Ind2 data. The proportion of fine particles (PM2.5) emitted by Ind1 was, in fact, considerably higher than that found for Ind2 (Félix, Almeida et al. 2013). Particle size does not hamper the EBC-Pb analysis, which translates exposure of different occupational settings (Félix, Almeida et al. 2013), although it may influence the assessment through B-Pb. The fact that finer particles may be easily absorbed into the blood through lungs (Snee et al. 1985; ATSDR 1999) offers a faster response of B-Pb to exposure and, thus, closer proximity to a linear correlation with EBC-Pb. Earlier studies reported on the rapid uptake of inhaled fine particles seem to corroborate this hypothesis (Chamberlain et al. 1975), suggesting that the absorption of Pb, present in the inhaled particulate matter, from the lungs into the blood circulation can be modulated by the relative abundance of coarse and fine particles in the environment (IPCS 1995). As the Pb concentration increased in the work environment, the increment of EBC-Pb was higher than in B-Pb. This seems to indicate that the retention of Pb in blood may slow down or decrease during exposure to high levels of Pb in PM, suggesting that B-Pb would be a biomarker more suitable for moderate and low levels of exposure. In these extreme conditions, the Pb concentration in blood may be influenced by limiting factors, such as, absorption and transport rates, and saturation of Pb-binding sites in erythrocytes and carrier proteins in serum (Fleming et al. 1997; Skerfving & Bergdahl 2007). Thus, the simultaneous determination of Pb in blood cells, serum and plasma fractions would help elucidate the absorption. The levels of interference in EBC-Pb and B-Pb are schematized in Figure 4. Exchanges between blood, organs and tissues are expectable. However blood–lung transitions are unlikely. Thus, EBC is not influenced by confounders and this is an advantage over blood in terms of a biomarker. EBC is apparently a matrix with less probability for bias than whole blood. In fact, a peak in B-Pb does not necessarily mean a strong and recent deposit in the lungs. On the other hand, a low B-Pb does not mean that the subject is not highly exposed at the moment (OSHA 1993; ATSDR 2007). Moreover, with the advancing age of exposed workers, and reduction in bone mass, the levels of B-Pb are potentially and increasingly biased by the release of Pb accumulated in bone (Silbergeld et al. 1988; O’Flaherty 1993; Gulson et al. 1996). The levels of B-Pb have a higher probability of being influenced by endogenous Pb. Presently, both EBC-Pb and B-Pb cannot indicate the dose of intake. However, by using EBC as a proxy to assess the particles settled in the airways, there is a high
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Figure 4. Schematic representation of the Pb mobilization pathways based on O’Flaherty (1993, 1995), including main routes of excretion (black arrows). Strikes over the arrow indicate an inexistent or negligible pathway.
potential to obtain dose information. Further studies should explore the capability of EBC to express the dose of intake by inhalation for an exposure period of interest. This would involve a follow-up of exposed individuals to different levels of Pb through inhalation, with periodic and simultaneous measurements of EBC-Pb and airborne Pb. Measurement of biomarkers of inflammation, including leukotriene B4 (Montuschi 2009) and metabolites (Montuschi et al. 2012; Motta et al. 2012), or 15-F2t-isoprostane, as a marker of oxidative stress, in EBC (Lucidi et al. 2008; Barreto et al. 2009; Montuschi et al. 2010) might be useful for assessing Pb pulmonary toxicity. Detecting breath VOC profiles by e-noses might also be a novel approach for identifying lung inflammatory changes (Fens et al. 2011; Bofan et al. 2013; Montuschi et al. 2013). Such data would be of utmost importance to model B-Pb changes and to quantify the contribution of sources of intake in blood. EBC proved to be useful for occupational exposure assessment and further trial research programmes would help establishing EBC as a complementary test in workers’ biomonitoring. The B-Pb is useful as a complementary tool to assess toxicity, given its ability to reflect systemic Pb, as in episodic endogenous Pb releases, which are factual risks, particularly on retired workers (Erkkilä et al. 1992; Gerhardsson et al. 1993). On the other hand, EBC-Pb is useful to assess Pb intake by inhalation and to estimate the burden of the target organ in continuously exposed subjects. The potential of EBC may offer a valid contribution to intake quantification, considering the amount of time the worker has been exposed to Pb. Hence, EBC may help predicting temporal limits of exposure and preventing long-term hazardous health outcomes, relatively common in chronic Pb exposure. Conclusions Ideally, the identification of exposure is done through a biomarker that reduces probable confounders and has the most reduced number of sources. The potential of EBC-Pb as a
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biomarker of exposure to Pb in occupational settings was emphasized in this study by (i) the ability of EBC to assess different levels of exposure; (ii) the minor influence of confounders, such as gender, age, smoking habits and working years; (iii) non-invasive characteristics, which allow a continuous monitoring of a subject by serial sampling; (iv) and analytically undemanding processes, resulting in expedite analysis and little sample manipulation. These features are important to validate EBC as a biomarker. Moreover, EBC opens the possibility to determine the dose by measuring the accumulation through time of a relevant exposure indicator and to assess variable exposures, such as work-post change. This makes EBC an attractive matrix in occupational exposure studies. Acknowledgements The authors gratefully acknowledge Fundação para a Ciência e Tecnologia (FCT) for funding the project PTDC/AMB/65828/2006 – Exhaled breath condensate: a tool for non-invasive evaluation of pollutant exposure? The authors thank Dr Marta Santos for the fruitful discussions and expertise in the ICP-MS.
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