Acute changes in sputum IL-10 following underground exposure to diesel exhaust

Abstract:

Although exposure to diesel exhaust has been linked with adverse health effects, little is known about the acute effects of exposure in the underground workplace. Methods. Cross-shift spirometry and sputum induction were completed on twelve subjects associated with comminuted rock removal (mucking) operations in an underground copper mine using diesel powered and pneumatic equipment on separate days, and sputum collected on a baseline non-exposure day as well. Results. For diesel operations, elemental carbon exposure averaged 538 ± 512 μg/m3 during the 1-2 hour operations. Sputum interleukin-10 decreased with diesel exhaust using one ELISA assay (3.69 v. 2.32 pg/ml, p = 0.015), but increased when measured with a different ELISA kit (0.18 v. 0.59 pg/ml, p = 0.019), consistent with an overall decline in IL-10 protein concentration but an increase in the biologically active form. Sputum interleukin-6 decreased with exposure to diesel exhaust, although this change lost statistical significance when restricted to non-smokers. There were no significant changes in spirometry, interleukins 1β, 4, and 8, tumor necrosis factor alpha or 8-hydroxy-2'-deoxyguanosine. 
Conclusion. High levels of diesel exhaust can result in rapid changes in sputum IL-10, suggesting possible protein modification.

Methods

The study was approved by the University of Arizona Institutional Review Board. Informed consent was obtained from all subjects volunteering to participate in the study. Mining students undergoing undergraduate and graduate training in mining engineering at the University of Arizona were eligible for participation.

The study was carried out at the San Xavier Mining Laboratory, a research and training faculty devoted to occupational health and safety in the mining and underground construction industries, operated under the auspices of the College of Engineering at the University of Arizona (UA), in collaboration with the UA College of Public Health and International Center for Mine Health, Safety, and Environment. 

While recognized as a copper deposit, the structure is a true polymetallic and contains a wide array of sulfide and oxide mineralization. The laboratory is comprised of three working levels employing both rubber-tired and rail equipment. Mine access and ventilation are designed to simulate underground conditions found in an actual production facility.

The subjects were evaluated for FEV1,  FVC, biomarkers in sputum, and respirable dust both before and during mucking operations. Two different types of muckers were used: 1) A diesel-powered 1984 Jarvis Clark JS-220 load-haul-dump (LHD) vehicle with a two cubic yard bucket and an 82 HP Deutz F6L-912W diesel engine fitted with a catalytic converter, and 2) a pneumatic (no emission) Eimco 12B Overshot Mucker, which served as an "activity control".

Respiratory protection was not worn during mucking operations. While the operating and exposure characteristics between these two types of muck- ing systems are radically different, inherent differences in physical exertion experienced between operators of these two mucking systems are considered minor and inconsequential. Experiments conducted using the LHD were employed in a conventional 4 m*4 m tunnel decline. A 15 hp axial auxiliary fan provided ventilation for the heading. Experiments utilizing the 12-B Overshot Mucker were conducted in 3 m*3 m drifts and employed the same type of ventilation circuit.

All subjects underwent three evaluations, generally at least one week apart, including a baseline non-exposure day, an overshot mucking day, and an LHD (diesel) mucking day. Sputum induction and a health history and exposure questionnaire were completed on non-exposure days. 

On mucking days, groups of 1-3 subjects first completed an interim health history and exposure questionnaire and underwent pre-shift spirometry testing, and then mucked for a 1-2 hour period using either an LHD unit or an overshot mucker. One hour following cessation of mucking, the subjects completed post- shift spirometry testing and sputum induction.

Industrial hygiene monitoring


Personal respirable dust sampling was performed on a limited number of subjects during LHD and overshot mucking, using SKC aluminum cyclones in the subjects' breathing zone attached to sampling pumps (SKC AirChek 2000, Eighty Four, PA).

 The pumps were calibrated at 2.5 SL/min, with an expected 50% cut size of 4.0 m. Pre- and post-sampling flow rates of the pumps were within 95%. Respirable dust sample measurements were collected on preweighed PVC filters and analyzed by gravimetric analysis (Cahn 21 Automatic Electrobalance, Ventron Corporation, CA). Gravimetric sampling results were corrected for changes in weight of field blanks collected each sampling day. Results from samples collected with sampling times less than 40 minutes in duration were not included in the calculation of exposure concentrations.

For diesel exhaust, samples were collected on precleaned 37 mm open-face quartz fiber filters (SKC, Eighty Four, PA) with MSA personal sampling pumps (Escort Elf, Pittsburgh, PA) set at 2.0 SL/min and analyzed for elemental carbon according to NIOSH method 5040 (16) by the Wisconsin
State Hygiene Laboratory (Madison, WI). Nitrogen dioxide and carbon monoxide concentrations were assessed with a MSA multigas detector (Mine Safety Appliance Company, Pittsburgh, PA) during a single mucking shift using the diesel powered LHD.

Spirometry testing/sputum induction and analysis

Pulmonary function testing was performed following American Thoracic Society (ATS) standards to evaluate changes in forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) (17). Spirometry was performed in sitting posture using a Puritan Bennett® electronic spirometer (Puritan Bennett, Lenexa, KS) immediately prior to mucking operations and again one hour following cessation of mucking. Induced sputum was collected using DeVilbiss Ultra-Neb 99HD ultrasonic nebulizers (Somerset, PA) filled with 3% saline set on maximum output. Sputum was collected follow- ing spirometry at baseline and one hour post-mucking for diesel and non-diesel operations. The sputum collection time period of one hour following cessation of exposure was based on the observed decrease in IL-10 one hour following cessation of overhaul exposure in firefighters (14).

Sputum samples were diluted with 10% Sputolysin® (Cal- biochem, San Diego, CA) in phosphate buffered saline with penicillin-streptomycin and 0.5% bovine serum albumin. Supernatant was removed by centrifugation and frozen to -80°C for later analysis of cytokines. The cellular pellet was reconstituted in 1 ml of the phosphate buffered saline in order to perform total cell counts with the use of a hemocytometer and Trypan Blue stain (Sigma Chemical CO, St. Louis). Our criteria for adequate sputum induction included a sputum cell count of 5*104 cells/ml following dilution with the 10% Sputolysin® solution.

A portion of the cell pellet was cytocentrifuged (Shandon Cytospin, ThermoShandon, Pittsburgh, PA) onto a micro- scope slide and stained with Diff-Quik® (Dade Behring AG, Switzerland) for cell differential analysis using the first 100 white cells counted and excluding epithelial cells. The super- natant was analyzed in duplicate for interleukin 1 beta (IL- 1β), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 8 (IL-8), IL-10, and tumor necrosis factor alpha (TNF-α) using commercially available ELISA kits (Biosource International, Camarillo, CA). Analysis for IL-4 and repeat analysis for IL- 10 was done using R&D ELISA plates (Minneapolis, MN). 8-hydroxy-2'-doxyguanosine (8-OHdG) levels in sputum were measured using a competitive ELISA commercial kit (8-OHdG Check, Japan institute for the Control of Aging, Fukuroi, Japan), following eightfold dilution with phosphate buffered saline.

Statistical analysis

Statistical analyses were performed using SPSS version 11.5 (Chicago, Illinois). The paired sample t-test was used to compare lung function data, which was normally distributed. The non- parametric Friedman's test for repeated measures data was used to compare cytokine and neutrophil measures pre- and post-mucking, and the Wilcoxon signed-rank test was used for post-hoc comparisons. Depending upon the distribution of the data, either Pearson's correlation coefficient or Spearman's rho was calculated.

Results

Twelve subjects, 11 males and one female, ranging in age from 19-33 (mean 23.7 ± 4.3 years), completed the study. Eight (67%) of the subjects described themselves as white, two (17%) as Hispanic, and one each as Asian and other. Only one subject reported current smoking (12 cigarettes/ day). None of the subjects were current asthmatics and no subjects reported taking anti-inflammatory medications. At baseline and prior to mucking, none of the subjects reported having had a cold, flu, allergies, or respiratory symptoms within the previous six days.

There was a minimum of 7 days between the three evaluations in all but two subjects, one of whom completed his overshot mucking evaluation four days following LHD mucking and one who completed his baseline non-exposure evaluation three days following overshot mucking.

The initial evaluation was LHD mucking for six subjects, a non-exposure day for five subjects, and overshot mucking for one subject. At non-exposure baseline testing, two subjects reported some low-level exposure to diesel exhaust, two reported some dust exposure, and none reported exposure to oil mist within the previous two days.

For overshot mucking, four subjects reported some low-level exposure to diesel exhaust, four subjects reported some dust exposure, and two subjects reported exposure to oil mist within the previous two days. For LHD mucking, two subjects reported some low-level exposure to diesel exhaust, one subject reported some dust exposure, and none reported exposure to oil mist within the previous two days.

Respirable dust sampling (n = 11) performed during overshot mucking demonstrated a mean of 0.50 ± 0.77 mg/m3 and a range of 0 to 1.52 mg/m3. Sampling times averaged 110 minutes (range 81-199 minutes). For LHD mucking, two respirable dust samples collected over a period of 66-68 min- utes demonstrated concentrations of less than 0.01 mg/m3. Elemental carbon exposure (n = 12) averaged 538 ± 512 (range 91-1800) μg/m3. Sampling times averaged 89 (range56-134) minutes. For a single LHD mucking shift monitored for 60 minutes, peak concentrations for NO2 and CO, respectively, were 1.5 ppm and 22 ppm.

There was a slight decline in FEV1 and FVC associated with LHD mucking; however, these changes were not statistically significant (p = 0.08 and 0.178, respectively) (Table 1). Exposure as measured by elemental carbon, duration of mucking, or the product of both variables was not significantly correlated with decline in FEV1.

For sputum IL-10, two different assay kits yielded different results with the same samples (Table 2). The R&D ELISA kit demonstrated a significant decrease in IL-10 after LHD muck- ing, compared with non-exposure days (p = 0.015). In contrast, using the Biosource IL-10 ELISA kit, there was a significant increase in IL-10 concentrations after LHD exposure com- pared with non-exposure days (p = 0.019 by Wilcoxon Signed Ranks test) (Fig. 1). These differences increased in statistical significance when smokers were excluded from the analysis.

There were significant differences in sputum IL-6 concentrations at baseline, post-overshot, and post-LHD mucking expo- sure (Friedman test, p = 0.046). In post-hoc comparisons, IL-6 concentrations were significantly lower after LHD mucking than at baseline (p = 0.034), but baseline and post-OS mucking IL-6 concentrations were not significantly different (p = 0.695). How- ever, when this analysis was done on non-smokers only, these differences lost statistical significance. TNF-α did not vary significantly among the non-exposure and mucking measurements. The percent of sputum white cells that were neutrophils did not change markedly with either form of mucking.

At baseline, the percentage of sputum neutrophils was positively correlated with age (r = 0.69, p = 0.014) (Fig. 2), but not with sputum IL-6, IL-8, or IL-10 concentration. Age was negatively correlated with sputum 8-OHdG.

