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Journal of Occupational & Environmental Medicine:
doi: 10.1097/JOM.0b013e3181570726
Original Articles

Clara Cell Protein as a Biomarker for Lung Epithelial Injury in Asphalt Workers

Ulvestad, Bente MD, PhD; Randem, Britt Grethe MD, MPH, PhD; Andersson, Lena MSc; Ellingsen, Dag G. MD, PhD; Barregard, Lars MD, PhD

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Author Information

From the Department of Occupational Medicine (Dr Ulvestad), Mesta AS, Moss, Norway; Department of Occupational Medicine (Dr Randem), Ulleval University Hospital, Oslo, Norway; Department of Occupational and Environmental Medicine (Ms Andersson, Dr Barregard), Sahlgrenska University Hospital and Academy, Goteborg University, Goteborg, Sweden; and National Institute of Occupational Health (Dr Ellingsen), Oslo, Norway.

CME Available for this Article at

This study was supported by a grant from the Statoil Research Fund for occupational research. The authors have no affiliations with companies or products that may be mentioned in this paper.

Address correspondence to: Bente Ulvestad, Department of Occupational Medicine, Mesta, AS, P. Box 5133, 1503 Moss, Norway; E-mail:

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Objective: We investigated if asphalt workers showed signs of lung epithelial injury as shown by increased Clara cell protein 16 (CC16) in serum after 6 months of exposure.

Methods: Asphalt pavers, asphalt plant operators, and asphalt engineers underwent lung function tests and blood samples before the start of the asphalt season. The tests were repeated before the end of the asphalt season. Blood samples were analyzed for concentration of CC16 and interleukin-6 (IL-6).

Results: After adjustment for current smoking, the pavers had a significantly larger increase in CC16 concentrations after the season as compared with that of the engineers and plant operators. In pavers, the change in serum CC16 was correlated with the change in IL-6.

Conclusion: CC16 increased over the season in pavers and appears to be a useful biomarker for lung epithelial injury in exposed workers.

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Learning Objectives

* List the types of exposure that have been reported in those working with asphalt–a mix of bitumen, crushed stone and gravel.

* Compare serum levels of Clara cell protein 16 (CC16) in asphalt workers, asphalt plant operators, and asphalt engineers before and at the end of the paving season, adjusting for smoking status and medication use.

* Identify associations, if any, between changes in serum CC16 over the asphalt paving season and changes in serum levels of interleukin-6 and 1-second forced expiratory volume, a measure of lung function.

The level of lung-specific proteins in serum has been proposed as a marker for lung epithelial injury following airway exposure to irritating agents. The motivation for this proposal is the idea that lung-specific proteins move passively across the epithelial barrier and into the blood to a larger extent when the barrier is damaged, and that these proteins may therefore serve as peripheral indicators of epithelial damage.1 Clara cell protein 16 (CC16) is a 16 kD anti-inflammatory protein secreted by the nonciliated bronchiolar Clara cells.2 Serum CC16 has been used as a biomarker in investigations of ozone-induced lung injury in humans,3 exposure to tobacco smoke,4 urban air pollution,5 an occupational group exposed to garbage and wastewater aerosols,6 firefighters exposed to smoke,7,8 and people exposed to chlorinated compounds in swimming pools9 or wood smoke (Barregard et al., submitted).

Asphalt work is seasonal work, carried out during the summer months in Nordic countries. Asphalt is a mix of bitumen with crushed stone/gravel. It is produced in asphalt plants by asphalt plant operators, and put down hot on the road by asphalt pavers. Asphalt work is associated with health risks from various exposures, including dust, oil mist, polycyclic aromatic hydrocarbons (PAHs), and nitrogen dioxide,10–13 and studies have reported enhanced mortality from respiratory diseases in asphalt workers. A standardized mortality ratio (SMR) of 207 (95% confidence interval [CI] = 95 to 393) from bronchitis, asthma, and emphysema in mastic asphalt workers was reported in 1991.10 An asphalt worker mortality study, involving 29,820 European asphalt workers, reported that, in comparison with construction workers, asphalt workers had a relative risk (RR) of 1.36 (95% CI = 1.06 to 1.74) of dying from non-malignant respiratory disease.11 In earlier research on asphalt workers (asphalt plant operators and asphalt pavers), we found that forced expiratory volume in one second (FEV1), and forced expiratory flow rate at 50% of the forced vital capacity (FEF50%) were significantly lower in the asphalt workers than in an external reference group of heavy construction workers. We also found a significant increase over the asphalt season in the mean plasma concentration of interleukin-6 (IL-6) in asphalt pavers.14 One group of asphalt pavers, the screedmen, who walk and work behind the asphalt paving machine, showed a statistically significant loss of forced vital capacity (FVC) and FEV1 over the season.

Our previous work, however, has not made it clear which exposure agents caused the observed lung function changes and IL-6 elevation. The paving team's exposure to total dust, respirable dust, total PAHs, and oil mist was low to moderate as defined by the Norwegian occupational exposure limits (OELs). The asphalt paving team had higher exposure to total PAHs than the asphalt plant operators had, but the exposure of the screedmen did not differ from the exposure of the rest of the asphalt paving team for total dust and total PAHs. It is possible that oil mist exposure differed between occupational groups, but we did not carry out a sufficient number of measurements to be able to assess this possibility.

The present report describes levels of serum CC16 in a group of employees partly overlapping that of our previous study14 (see below for details). The new data presented here includes a reanalysis of the serum samples collected in 2005, and the results of new lung function tests in the pavers, performed prior to the asphalt paving season of 2006.

We hypothesized 1) that the workers of the asphalt paving team would have a higher level of CC16 in serum at the end of the work season than before the work season, and that this change would be smaller or absent in the less-exposed internal control groups of asphalt plant operators and asphalt engineers, and 2) that the differences in CC16 response would be associated with lung function changes and changes in IL-6.

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Materials and Methods


All the asphalt pavers (hereafter called pavers), a group of asphalt plant operators (hereafter called plant operators), and a group of asphalt engineers (hereafter called engineers) employed in Norway's largest road construction and road maintenance company underwent lung function tests and blood samples in April/May 2005, prior to the start of the asphalt season. Of the 184 subjects tested before the season, 49 were contract workers who left the company shortly after the first medical tests, and an additional 12 (nine pavers, two plant operators, and one engineer) were lost to follow-up. Lung function tests and blood samples (including reanalyses of frozen serum samples) were repeated in the remaining 123 subjects (72 pavers, 32 plant operators, and 19 engineers) just before the end of the asphalt season, in September/October 2005. Demographic data of these 123 study subjects at the start of the study are given in Table 1. Lung function tests were again administered before a new asphalt season started, in April/May 2006, but due to study constraints only the pavers participated in this round of tests. The subjects partly overlapped those of the previous study.14 However, we excluded lorry drivers, since they may have had other exposures, and instead included the engineers, who were considered to have very low-grade exposure, and so could be used as a reference group. All subjects were employed by the same company and were examined in the same manner before and after the asphalt season.

Table 1
Table 1
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The National Data Inspectorate and the Regional Medical Board of Ethics approved the study. All participants provided informed written consent.

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Exposure Assessment
Sampling strategy.

Measurements were carried out between April and October 2005 and between April and October 2006. A random sample of workers, representing different work tasks, were asked to participate in the exposure assessment. Participation was voluntary, but all the selected workers agreed to participate.

Exposure to dust and gases was determined by personal sampling, and two or more types of measurements (see below) were performed simultaneously for 7 to 8 hours for each person for at least 2 consecutive days.

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Sampling Methods and Analyses

Total dust and particulate polycyclic aromatic hydrocarbons (PAHs) were collected on glass fiber filters (Whatman International Ltd, Maidstone, UK), fitted in 37-mm closed-faced aerosol filter cassettes (Millipore Corporation, Bedford, MA). Gaseous PAHs were collected on tubes filled with the adsorbent XAD-2 (SKC, Blandford Forum, UK). Filters and tubes for collection of particulate and gaseous PAHs were mounted in series during sampling. The sampling flow rate was 2 L/min. The filters and adsorbents were extracted by addition of dichloromethane mixed with internal standards (Naphthalen-D8, Phenanthren-D10, Fluoranthen-D10, Benzo (a) pyren-D12).

Particle mass was measured using a microbalance (Metler Toledo AT261, Columbus, OH), with a detection limit (three times the SD of a blank filter) of 0.031 mg/m3 based on 8 hours of sampling. Total PAHs were analyzed by use of gas chromatography (GC) with a mass selective detector (MS) (Agilent, GC/MS 6890, Santa Clara, CA). The detection limits of the PAHs were 0.005 to 0.010 μg/m3 depending on the specific component, based on 8 hours of sampling at a flow-rate of 2 L/min.

Oil mist was collected on glass fiber filters (Whatman GF, Madistone, UK), and oil vapor was collected on tubes containing XAD-2 (SKC, Blandford Forum, Dorset, UK). Filters and tubes for collection of oil mist and oil vapor were mounted in series during sampling. The sampling flow rate was 2 L/min. Oil mist was determined using a Fourier transform (FT-IR) spectrophotometer, PE-1600 (Perkin Elmer, Waltham, MA), and oil vapor by chromatography (Agilent, GC/MS 5890, Santa Clara, CA) with a flame ionisation detector (FID). The detection limits of oil mist and oil vapor were 10 μg/m3 and 5 μg/m3, respectively, based on 8 hours of sampling at a flow rate of 2 L/min.

Carbon monoxide and nitrogen dioxide concentrations were measured with direct-reading electrochemical sensors with a data-logging facility built into the instrument (type PAC III, Dräger AG, Lübeck, Germany). An averaging period of one reading every 2 minutes was selected. The detection limits of carbon monoxide and nitrogen dioxide measurements were 2 ppm and 0.2 ppm, respectively.

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Assessment of Respiratory Health Effects
Lung function assessments.

The first lung function tests were performed prior to the start of the 2005 asphalt season, between 7 and 9 am, in pavers, plant operators, and engineers. A few days before the asphalt season ended in October 2005, lung function tests were performed again at the same time of day. New lung function tests were carried out among the pavers approximately 6 months later, prior to the start of the new asphalt season in April/May 2006.

Lung function tests (FVC, FEV1, and FEF50%) were carried out using bi-directional ultrasound transit time measurements with a Spirare SPS310 spirometer, in accordance with the guidelines recommended by the American Thoracic Society.15 The lung function variables were expressed in absolute values and as percentages of the predicted value according to the reference values of the European Coal and Steel Community (ECSC).16

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Information on age and smoking status was obtained from a general questionnaire used in an earlier cross-sectional study.13 The workers answered the questionnaire before the examinations. Subjects were classified as lifelong non-smokers, former smokers, and current smokers. Former smokers were those who had stopped smoking more than 12 months earlier.

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Assessment of Inflammatory Responses

Blood samples for the analysis of CC16 and IL-6 were collected between 7 and 9 am just before the start and just before the end of the 2005 asphalt season, at the same time as the lung function tests. Plasma samples were obtained using EDTA tubes, kept on ice for up to 60 minutes before they were centrifuged at 2000g for 15 minutes. Serum samples were obtained after keeping blood samples at room temperature for coagulation for 60 to 120 minutes before they were centrifuged at 1300 g for 15 minutes. Plasma and serum were then transferred into cryo tubes, frozen on dry ice, and transported to the Ulleval University Hospital, where they were stored at –70°C. A year later, frozen serum on dry ice was transported to Goteborg (see below).

CC16 was analyzed at the Department of Occupational and Environmental Medicine, Sahlgrenska University Hospital and Academy, Goteborg, using the human Clara Cell Protein ELISA kit from BioVendor (BioVendor Laboratory Medicine Inc., Brno, Czech Republic), as previously described.17 Plasma samples were analyzed for IL-6 concentration at the Centre for Clinical Research, Ulleval University Hospital of Oslo, Norway, using a commercial ELISA kit (Quantikine HS from R & D systems, Abingdon, UK). The sensitivity of the assay is 0.039 pg/mL. For both CC16 and IL-6, before-season and after-season samples were analyzed simultaneously without knowledge of identity code, exposure status, or time.

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Data Analysis

With the use of cumulative probability plots, the exposure data were found to be best described by log-normal distributions, and were therefore ln-transformed before further statistical analyses. The measured exposure variables were used without further adjustment as they were regarded as representative of the whole work shift. Standard measures of central tendency and distributions (geometric means [GM] and geometric standard deviations [GSD]) were calculated. Mann-Whitney tests were used for two-group comparisons for total dust, total PAHs, and oil mist. IL-6 was also ln-transformed before statistical analyses. The concentration of CC16 and the lung function parameters showed normal distributions.

The relationships between the changes in CC16 and job group and smoking category were tested by analysis of variance (ANOVA). Analyses were repeated without subjects who reported use of asthma medication. The associations between the changes in FEV1 and in ln-transformed IL-6 over the asphalt season were examined using the Pearson correlation coefficient. Statistical analyses were carried out using SPSS, version 14.0 (SPSS Inc, Chicago, IL).

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The engineers were somewhat older than the pavers and the plant operators were, but the groups were comparable with respect to both body mass index (BMI) and smoking habits (Table 1). The groups were also comparable with respect to FEV1 (percent of predicted) before the asphalt season started in 2005 (Table 1). Six of the plant operators were former pavers, and seven of the engineers were former asphalt workers.

The plant operators had significantly higher exposure to total dust than the pavers had, while the pavers had significantly higher exposure to total PAHs than the plant operators had (Table 2). Although the geometric mean concentration of oil mist was less than 0.3 mg/m3, the exposure to oil mist was significant on some occasions; for example, on one occasion a level of 1.7 mg/m3 was measured in an asphalt paving machine without a cabin. The geometric mean concentration of nitrogen dioxide in pavers was less than 1 ppm. The pavers may, however, have been exposed to higher levels when paving asphalt in tunnels (max level measured was 3.4 ppm). Only a few analyses of respirable dust, volatile organic compounds, and carbon monoxide were carried out. The engineers were judged not to be exposed to air pollution in their current work.

Table 2
Table 2
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The mean loss in FEV1 over the asphalt season was only 0.017 L, and it was not statistically significant in any of the three groups. It was, however significant in smokers (N = 30, mean loss 0.14 L, P < 0.0001). Non-smoking pavers showed a significant loss in FEV1 1 year later (FEV1 decrease = 0.072 L; standard deviation [SD] = 0.19, P = 0.03). Smoking pavers showed a loss of FEV1 after 1 year (FEV1 decrease = 0.101 L), which was not statistically significant (P = 0.1).

Table 3 shows the serum CC16 concentrations before the start of the asphalt season and the change in CC16 during the season. In the entire study population, smokers showed a statistically significantly lower level of CC16 than non-smokers did before the season, but had a larger increase in CC16 during the season. There were no differences between job groups in CC16 concentrations before the season, but the pavers had a significantly larger increase in CC16 concentrations after the season compared to that of the engineers, when adjusted for current smoking. The increase was also larger than that of the combined group of plant operators and engineers, with a borderline statistical significance (P = 0.05). The larger change in CC16 over the season was even more prominent when the comparison was restricted to non-smokers, as illustrated in Fig. 1 (pavers vs engineers P = 0.003, pavers vs combined group P = 0.02). Analyses were repeated and found to be unchanged after exclusion of seven subjects on asthma medication.

Table 3
Table 3
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Fig. 1
Fig. 1
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In pavers, the change in serum CC16 over the asphalt season was correlated to the change in ln-transformed IL-6 (r = 0.25, P = 0.04). There was no consistent association between the change in serum CC16 and the change of FEV1 over the asphalt season.

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In this study of asphalt workers, we investigated the effects of exposure in asphalt work on changes in CC16 and lung function. Our main finding was an increase in CC16 over the asphalt season in pavers compared both with that of engineers and with that of the combined group of engineers and plant operators. The plant operators and the engineers also showed a small CC16 increase over the asphalt season, but it was not statistically significant. In pavers, the increase of the serum level of CC16 was correlated to the increase of the level of IL-6.

Our internal reference groups consisted of asphalt plant operators and asphalt engineers; the plant operators were also exposed, but to a lesser extent than the pavers were. The educational and occupational background and the proportion of current smokers were similar in the two groups of blue-collar workers, while the proportion of current smokers was somewhat lower among the engineers. Potential confounders, such as age, body mass index, and reported allergy, did not change the main results.

The increase in CC16 could not have been due to diurnal variation, because all blood samples were drawn between 7 and 9 am for all subjects both before and after the season. The serum analyses were carried out after about 12 months and 18 months of storage at –70°C. The engineers showed a small and statistically non-significant increase in CC16 concentrations over the season, which may possibly be explained by chance or by longer storage of the before-season serum samples. However, such an effect could not explain the more pronounced increase in the pavers.

Asphalt workers are exposed to a variety of exposure factors, and it is still not clear which specific agents caused the observed increase in CC16. Plant operators spend most of the working day inside their well-ventilated cabins, controlling the asphalt production via advanced information technology, but they have to leave their cabins several times during the day for adjustments. Leaving the cabin, they become exposed to dust from gravel, usually in low concentrations. The bitumen and stone/gravel that constitute the asphalt mass are mixed under closed conditions in the asphalt plant, so exposure to oil mist is probably very low for the plant operators. The pavers have a lower exposure to total dust than the plant operators do, but most of their exposure is oil mist, with other biological properties. The occurrence of obstructive lung disease in workers exposed to oil mist was described as early as 1988.18 It is possible that the PAHs or irritant VOCs in the asphalt fumes are important. We found low concentrations of nitrogen oxides, and this probably implies that diesel exhaust plays a minor role in the pavers' exposure, except when paving in tunnels.

Among the pavers (smokers and non-smokers together), we found a mean increase of 2.8 μg/L in the concentration of CC16. This is similar to results found in healthy subjects 2 to 4 hours after ozone exposure.3 In that study, levels of CC16 were back to baseline levels 18 hours after exposure. However, in a study of experimental exposure to wood smoke (Barregard et al., submitted), the change in serum CC16 was highest on the morning after exposure. In the present study, the blood samples taken at the end of the season were drawn in the morning, 12 to 16 hours after the last day's exposure. It is unclear whether the increase of serum CC16 in pavers reflects a temporary (over the day) effect on the air-blood lung epithelial barrier or a subchronic (months-long) effect.

Before the asphalt season, the smokers in the whole study population had significantly lower concentrations of CC16 in serum than the non-smokers did, a result in accordance with earlier studies.4,5,19,20 One theory is that chronic tobacco smoke exposure leads to depletion of Clara cells in the bronchial epithelium. However, even though the smokers had lower baseline values, they had the highest increase in CC16 serum levels during the season. This may reflect the effect of the combined exposure of tobacco smoke and bitumen fume. Non-smoking pavers, exposed for 13.1 (SD = 8.7) years on average, did not have lower serum CC16 before the season than non-smoking engineers had. This speaks against a depletion of Clara cells by long-term paving work.

In conclusion, serum CC16 seems to be a useful biomarker for lung epithelial injury in exposed workers. Further studies are needed to elucidate which exposure agents are responsible for this effect.

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1. Hermans C, Bernard A. Lung epithelium-specific proteins: characteristics and potential applications as markers. Am J Respir Crit Care Med. 1999;159:646–678.

2. Broeckaert F, Bernard A. Clara cell secretory protein (CC16): characteristics and perspectives as lung peripheral biomarker. Clin Exp Allergy. 2000;30:469–475.

3. Blomberg A, Mudway I, Svensson M, et al. Clara cell protein as a biomarker for ozone-induced lung injury in humans. Eur Respir J. 2003;22:833–888.

4. Robin M, Dong P, Hermans C, Bernard A, Bersten AD, Doyle IR. Serum levels of CC16, SP-A and SP-B reflect tobacco-smoke exposure in asymptomatic subjects. Eur Respire J. 2002;20:1152–11561.

5. Berthoin K, Broeckart F, Robin M, Haufroid V, De Burbure C, Bernard A. Serum pneumoproteins and biomarkers of exposure to urban air pollution: a cross-sectional comparison of policemen and foresters. Biomarkers. 2004;9:341–352.

6. Steiner D, Jeggli S, Tschopp, et al. Clara cell protein and surfactant protein B in garbage collectors and in wastewater workers exposed to bioaerosols. Int Arch Occup Environ Health. 2005;78:189–197.

7. Bernard A, Hermans C, Van Houte G. Transient increase of serum Clara cell protein (CC16) after exposure to smoke. Occup Environ Med. 1997;54:63–65.

8. Burgess JL, Nanson CJ, Bolstad-Johnson DM, et al. Adverse respiratory effects following overhaul in firefighters. J Occup Environ Med. 2001;43:467–473.

9. Carbonelle S, Francaux M, Doyle I, et al. Changes of serum pneumoproteins caused by short-term exposures to nitrogen trichloride in indoor chlorinated pools. Biomarkers. 2002;7:464–478.

10. Hansen ES. Mortality of mastic asphalt workers. Scand J Work Environ Health. 1991;17:20–24.

11. Boffetta P, Burstyn I, Partanen T, et al. IARC Epidemiological Study of Cancer Mortality Among European Asphalt Workers. Final Report. Lyon, France: International Agency for Research on Cancer; 2001.

12. Norseth T, Waage J, Dale I. Acute effects and exposure to organic compounds in road maintenance workers exposed to asphalt. Am J Ind Med. 1991;20:737–744.

13. Randem BG, Ulvestad B, Burstyn I, Kongerud J. Respiratory symptoms and airflow limitation in asphalt workers. Occup Environ Med. 2004;61:367–369.

14. Ulvestad B, Randem BG, Hetland S, Sigurdardottir G, Johannessen E, Lyberg T. Exposure, lung function decline and systemic inflammatory response in asphalt workers. Scand J Work Environ Health. 2007;33:114–122.

15. American Thoracic Society. Standardization of spirometry-1987 update. Am Rev Respir Dis. 1987;136:1285–1298.

16. Quanjar PH, Tammeling GJ, Cotes JE. Standardized lung function testing. Eur Respir J. 1993;10:1683–1693.

17. Andersson L, Lundberg P-A, Barregard L. Methodological aspects on measurement of Clara cell protein in urine as a biomarker for airway toxicity, compared to serum levels. J Appl Toxicol. 2007;27:60–66.

18. Robertson AS, Weir DC, Burge PS. Occupational asthma due to oil mists. Thorax. 1988;43:200–205.

19. Backe E, Lotz G, Tittelbach U, Plitzko S, Gierke E, Schneider D. Immunological biomarkers in salt miners exposed to salt dust, diesel exhaust and nitrogen oxides. Int Arch Environ Health. 2004;77:319–327.

20. Bernard AM, Roels HA, Buchet JP, Lauwerys RR. Serum Clara cell protein: an indicator of bronchial cell dysfunction caused by tobacco smoking. Environ Res. 1994;66:96–104.

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