Airway inflammation in iron ore miners exposed to dust and diesel exhaust

ABSTRACT:

The aim of the present study was to investigate if underground miners exposed to dust and diesel exhaust in an iron ore mine would show signs of airway inflammation as reflected in induced sputum.

In total, 22 miners were studied, once after a holiday of at least 2 weeks and the second time after 3 months of regular work. Control subjects were 21 "white-collar" workers. 

All subjects completed a questionnaire regarding medical and occupational history, and underwent lung function testing and induced sputum collection. 

Total and differential cell counts and analyses of the fluid phase of the induced sputum were performed. Sampling of personal exposure to elemental carbon, nitrogen dioxide and inhalable dust was recorded.

The average concentrations of inhalable dust, nitrogen dioxide and elemental carbon were 3.2 mg.m-3, 0.28 mg.m-3 and 27 mg.m-3, respectively. 

Miners had increased numbers of inflammatory cells, mainly alveolar macrophages and neutrophils, and increased concentrations of fibronectin, metalloproteinase-9 and interleukin-10 in induced sputum compared with controls.

In conclusion, miners in an underground iron ore mine demonstrated persistent airway inflammation that was as pronounced after a 4-week holiday as after a 3-month period of work underground in the mine.

METHODS

The mining environment

The present study was carried out in a Swedish iron ore mine, where all mining since the 1920s has been carried out underground. The dominant mode of mining is sublevel caving. Diesel-powered engines were introduced into the mine in the mid 1960s.

The air pollution in the investigated mine was complex. The most important sources include diesel exhaust and rock dust, which are vortexed into the air by the traffic in the mine, and/ or dust drilling, loading and crushing of the rock/ore.

Blasting routinely happens at 00:00 h. The mine is then ventilated until 05:00 h, at which point the miners can enter the mining areas if the levels of carbon monoxide and nitrogen dioxide are satisfactory. However, these blasting gases are also released, to some extent, during the handling of the blasted rock and ore.

Subjects

Healthy underground miners were invited to take part in the study. Eligible subjects were identified from the company health-service registry according to the following entry criteria: 1) no history of respiratory disorder, including bronchitis or any other respiratory symptoms; 

2) never-smokers or ex-smokers for <5 yrs; 3) no history of eye and/or nose allergies; 4) no treatment with anti-inflammatory drugs for any condition; 5) underground work in the mine for >3 yrs; and 6) a work site underground with exposure to diesel exhaust during loading, lorry driving, road maintenance or construction work.
 
Control subjects

Healthy nonsmoking workers (research workers not affiliated with the study, technical staff and office workers), without a history of respiratory conditions, allergies and other disorders and without any relevant medications were invited to participate as control subjects.

All subjects received both verbal and written information about the study and consented to participation. The Ethics Committee of Umea University (Umea, Sweden) approved the study.

Study design

The miners were investigated twice. The first examination took place after >4 weeks of a summer holiday in August, and before returning to the mine. This was to investigate subjects under conditions free of recent occupational exposure to diesel exhaust or dust.

The second examination was performed in November after >3 months of regular work in the mine. The investigation included a thorough occupational history pertaining to exposure to dust and diesel exhaust, as well as previous disorders focusing on respiratory conditions and allergies. No respiratory infection within the last 4 weeks of each examination was allowed.

Lung function

Lung function measurements were performed prior to a sputum induction. The control subjects were studied at a single time point during winter months. Three reproducible measurements of forced vital capacity and forced expiratory volume in one second (FEV1) were performed and the best value recorded. The values were expressed as per cent of predicted normal using the reference values from the Coal and Steel Union.

Sputum induction

Sputum induction was performed by a method described by PIN et al., which was slightly modified. Hypertonic saline (4.5%) was nebulised using an ultrasonic nebuliser (DeVilbis 2000; DeVilbis Co, Somerset, PA, USA) with an output of ~1.5 mL.min-1.

All subjects were pre-treated with an inhaled b2-agonist (0.2 mg salbutamol) before the induction. Inhalation of the hypertonic saline was made at three intervals of 5 min. FEV1 was monitored before and after each inhalation period. Following each inhalation period, subjects were advised to blow their nose and rinse their mouth with water before coughing sputum into a sterile container. The samples obtained were kept on ice for up to 1 h before processing.

Sputum processing

Sputum was processed according to the method described by PIZZICHINI et al.
At least 400 nonsquamous cells were counted and differential cell counts were expressed as percentage of the total nonsquamous cell count. The proportion of squamous cells was obtained by counting 400 additional cells and expressing this as a percentage of the total number of cells. 

Samples were considered adequate for analysis if the squamous cell contamination was <20% and the viability >50%. The total cell count was calculated by dividing the total number of cells by the volume of processed sputum (1 mg=1 mL).

Fluid-phase measurements

Fluid-phase measurements of matrix metalloproteinase (MMP)-9 and fibronectin reflect the activity of alveolar macrophages. Myeloperoxidase (MPO) is an oxidising enzyme whose levels reflect neutrophil activation. Interleukin (IL)-10 is an inflammatory cytokine with predominantly inhibitory actions and is an important modulator of monocyte/macrophage function.

Myeloperoxidase

MPO was analysed using a radioimmunoassay (Pharmacia MPO RIA; Pharmacia & Upjohn Diagnostics Sverige AB, Uppsala, Sweden). The detection limit of this assay was,8 mg.L-1.

IL-10, MMP-9 and fibronectin

IL-10, MMP-9 and fibronectin were measured by ELISA technique (IL-10 and MMP-9 kits from R&D systems Europe Ltd, Abingdon, UK, and fibronectin from Pharmacia & Upjohn Diagnostics). The minimum detection limits for IL-10, MMP-9 and fibronectin were <0.5 pg.mL-1, <0.156 ng.mL-1 and 10 mg.L-1, respectively.

Lung function

Lung function measurements were made using a dry bellows spirometer (Vitalograph Ltd, Buckingham, UK).

Air sampling

Elemental carbon (EC), nitrogen dioxide and inhalable dust were measured using sampling equipment attached to the subjects and measurements obtained in the breathing zone of the workers. Two measurements of EC and nitrogen dioxide, and three measurements of inhalable dust were obtained for each miner. 

Occupational exposures are often quasi lognormally distributed; that is, the log-transformed concentrations follow the normal distribution. By multiple measurements of each worker, the total variance in a group of workers can be divided by within-worker (day to day) and between-worker (within jobs) variance.

A uniformly exposed group characterised by a small between-worker variance is sometimes measured by the ratio (BR95) of the 97.5th to the 2.5th percentiles of the means. A group with a ratio .2 is often considered to be uniformly exposed.

Elemental carbon

EC was sampled on a glass-fibre filter (SIMPEDS, Safety In Mines Personal Equipment for Dust Sampling; Casella Ltd, Bedford, UK) and the amount was determined by colorometric analysis. The colorometric analysis was performed according to standard VDI 2465 at a Swiss laboratory (Institute Universitaire Romand de Santeau Travail, Lausanne, Switzerland).

Inhalable dust

The amount of inhalable dust was measured with an IOM personal sampler (SKC Inc., Eighty Four, PA, USA) and sampled according to the standard EN 481.

This samples the fraction of particles that enter the nose and mouth with a 50% cut-off point of particles with a diameter of 100 mm. The sampling flow was 1.7-2.1 L.min-1. The amount of dust was determined by weighing the filters.

Nitrogen dioxide

Nitrogen dioxide was measured using personal diffusive samplers and ion chromatographic analysis.

Statistics

Empirically, occupational exposures are usually log-normally distributed and the Shapiro-Wilk test was used to test the hypothesis of log-normal distributions. Paired t-tests were used to compare lung function measurements in miners between investigations. 

Independent sample t-tests were used for comparisons between subjects and controls. Sputum cell counts and fluid-phase measurements are given as median and interquartile range.

For the fluid-phase measurements, the Wilcoxon test was used for comparisons in miners between investigations, and a Mann–WhitneyU-test was used for comparisons between miners and controls.A p-value <0.05 was considered statistically significant

RESULTS

Demographics and lung function

The initial study population comprised of 29 male miners. However, six miners were excluded from all analyses due to the inability to produce sputum of sufficient quality for the cell analyses, and one was unable to complete both investigations. 

Therefore, data are presented for a total of 22 miners from whom paired sputum samples were available. The mean (range) age was 43 (30-59) yrs and, whilst all were current nonsmokers, 10 were never-smokers and 12 ex-smokers. Of the ex-smokers, six had been regular smokers (five had been smoke free for >10 yrs, and one had been free of smoking for at least 6 yrs) and six were previous occasional smokers.

Five of the regular ex-smokers had 10-12 pack-yrs and one had a 6pack-yr history of smoking. None of the subjects had a history of chronic bronchitis. The mean (range) number of years spent working underground was 18 (3-39) yrs.

The control group comprised of 24 male researchers, technical staff members or office workers without allergies or history of respiratory disease or other disorders. All but one were neversmokers. In the control group, the sputum samples were of insufficient quality to be analysed further in three cases. 

Thus, data from 21 control subjects are reported. The mean (range) age was 40 (25-63) yrs. All subjects, both miners and controls had normal lung function measurements (table 1). No significant differences in lung function were seen between miners and controls.

Lung function measurements in table 1 are from the first assessment of the miners after the holiday. There was no difference in the lung function of the miners between the two test occasions.

Sputum cell findings

The cell viability in sputum was good, with a mean per cent viability of 79% in miners and 78% in control subjects. There were statistically significant differences in the total number of cells.mL-1 of sputum between miners and controls.

This was mainly due to increased numbers of macrophages and neutrophils in the miners. As shown in table 2, there were no significant differences in numbers of lymphocytes or eosinophils between groups.

The total cell numbers and the differential cell counts were not statistically different between the first and the second sputum induction in the miners (table 3).

Fluid-phase measurements

Levels of MPO measured in sputum from miners on both occasions and controls did not differ. Fibronectin levels and IL10 were elevated in sputum from miners from both inductions compared with control subjects, but there was no difference between assessments.

MMP-9 levels were greater in miners than controls, but were reduced following a 3-month period at work (table 4).

Exposure

Nitrogen dioxide

In total, 29 samples from 18 individuals were collected. For 11 miners, the measurements were made on two occasions. The mean (range) was 0.28 (0.05-0.68) mg?m-3. 

Elemental carbon

In total, 27 measurements were made for 18 miners, and in eight individuals the measurements were made on two separate occasions. The mean (range) for all measurements was 27 (5–61)mg?m-3.

Inhalable dust

From 14 miners, 41 measurements were obtained. For all individuals, two or three measurements were made on separate occasions. A single high value of 35 mg?m-3 was included in the analysis. The second highest value was 9.3 mg?m-3. The mean (range) dust level was 3.2 (0.1-35) mg?m-3.

For all exposures, the hypothesis of log-normal distributions could not be rejected. For nitrogen dioxide and EC, the variations within miners were higher than between miners. The ratios between the 97.5th and the 2.5th percentile of the log-normally distributed mean exposures of the miners (between-worker distribution), equivalent to a factor containing 95% of the individual mean exposures derived from lognormal distribution, were 1.2, 1.6 and 5.0 for nitrogen dioxide, EC and inhalable dust, respectively (table 5).
 

 
