Abstract
Exposure to synthetic fibers with employment in textile mills can be associated with an elevated risk of interstitial lung disease (ILD). A mechanism of injury has not been determined. ILD can follow exposures to inorganic fibers (e.g., asbestos) which are associated with a mobilization of iron and catalysis of an oxidative stress. We describe 2 patients with ILD associated with exposure to synthetic textile fibers who demonstrated carbon-based ferruginous bodies suggesting an in vivo accumulation of iron by synthetic fibers after deposition in the lung. These iron-laden bodies varied from perfectly linear fibers to almost particulate matter. Linear structures were irregularly interrupted by deposition of iron-abundant material. The capacity of these synthetic fibers to complex iron and generate an oxidative stress is confirmed in vitro.
Introduction
Exposure to synthetic fibers with employment in a textile mill can be associated with an elevated risk of interstitial lung disease (Pimentel et al., 1975; Lougheed et al., 1995; Kern et al., 1998; Eschenbacher et al., 1999). A spectrum of histopathology has been described in textile workers with interstitial injury and this can include findings consistent with diffuse interstitial pneumonitis, diffuse alveolar damage, and bronchiectasis (Pimentel et al., 1975; Lougheed et al., 1995). Workers in the nylon flocking industry have more recently been described to be at an increased risk for chronic interstitial lung disease designated Flock Worker’s Lung (FWL) (Kern et al.,1998, 2000; Boag et al., 1999; Burkhart et al., 1999; Eschenbacher et al., 1999; Kuschner, 2000; Washko et al., 2000; Barroso et al., 2002). Pathologically, FWL was first characterized by bronchiolocentric nodular and diffuse lymphocytic interstitial infiltrates, a lymphocytic bronchiolitis, and variable interstitial fibrosis. Later, any pathologic evidence of interstitial lung disease would be accepted as supporting the diagnosis of FWL (Kern et al., 2000; Kuschner, 2000). Granulomas and giant cells have not been described.
A mechanism for these lower respiratory tract injuries associated with textile work has not been determined. Interstitial lung disease can follow chronic exposures to inorganic fibers (e.g., asbestos). Inflammation and fibrosis after asbestos inhalation correlate with a mobilization of iron from either the fiber matrix itself or the host (Lund and Aust, 1991). Some portion of this metal is catalytically active and an oxidative stress is produced, transcription factor activation and release of mediators follow, and inflammation and fibrosis result. Synthetic fibers do not include metals. However, following deposition in the lung, synthetic fibers present an insoluble solid-liquid interface to the surrounding host tissue. Comparable to the inorganic fibers, the surface of the synthetic fiber will include functional groups with a capacity to mobilize metal from endogenous sources. Therefore, the deposition of a synthetic fiber could introduce a metal-catalyzed oxidative stress in the lower respiratory tract similar to that of inorganic fibers. The ferruginous body, with its accumulated iron, functions as an indicator of both metal accumulation and a potential oxidative stress. Ferruginous bodies have not been found in the human lung following exposure to synthetic fibers. We describe 2 patients with interstitial lung disease associated with exposure to synthetic textile fibers who demonstrated ferruginous bodies suggesting an accumulation of iron onto the fiber after its deposition in the lung. The capacity of synthetic fibers to complex iron and generate an oxidative stress is then confirmed in vitro.
Materials and Methods
Electron microscopy of fibers collected from the textile mill
A dust sample was collected by hand from several surfaces in the textile mills by simply acquiring it from the top of factory machinery. Fibers were examined using SEM and EDXA.
In Vitro Iron Uptake by Fibers
Ten mg of fiber was added to 10.0 ml of either saline or 1.0 mM FeCl3 in saline and agitated for 60 minutes. After washing, surface iron was measured in triplicate by treating the dusts with 2% HNO3 and quantifying the metal concentrations in the supernatant using a Perkin Elmer Model P40 ICP/AE spectrometer at a wavelength of 238.204 nm.
In Vitro Oxidant Generation
Generation of oxidants by fibers was measured using thiobarbituric acid (TBA) reactive products of deoxyribose. The pentose sugar, 2-deoxy-D-ribose, reacts with oxidants to form products which, after heating with TBA at a low pH, produces a pink chromogen quantified by measuring absorbance at 532 nm. Reaction mixtures contained 1.0 mM deoxyribose, 1.0 mM H2O2, 1.0 mM ascorbate, and the fiber 1.0 mg/ml. The suspension was incubated at 37°C for 4 hours with agitation and then centrifuged at 1200 g for 10 minutes. One ml of both 1.0% (w/v) TBA and 2.8% (w/v) trichloroacetic acid were added to 1.0 ml of supernatant, heated at 100°C for 10 minutes, and cooled in ice. Assays of oxidant generation were repeated with either a hydroxyl radical scavenger, dimethylthiourea (DMTU), or an iron chelator, deferoxamine, added to the reaction mixture (final concentration 1000 μM). The absorbance of the chromophore was read at 532 nm on a Beckman DU 640B spectrometer.
Case Reports
Patient 1
A 56-year-old African-American female was referred with the only symptom being dyspnea on exertion. She was a lifetime nonsmoker. Her job for the last 20 years was grading and inspecting in a textile mill that used Nylon and Rayon in the production of both men’s suits and fabric for office walls. There was no report of any employment allowing exposure to asbestos or silica. Inspiratory crackles were noted on examination. Pulmonary function tests demonstrated a forced vital capacity (FVC) of 1.35 L (51% predicted), a forced expiratory volume in 1 second (FEV1) of 1.16 L (55% predicted), and a FEV1/FVC of 86%. The total lung capacity (TLC) was 2.41 L (55% predicted) and the diffusion capacity was 8.0 mL/minute/mm Hg (37% predicted). An interstitial pattern was evident on the chest X-ray. The high resolution CT scan of the chest (supine and prone) exhibited subpleural interstitial thickening involving all lung zones (Figure 1). A thoracoscopic biopsy revealed a diffuse and temporally variegated pattern of interstitial fibrosis with honeycomb formation and mucopurulent material. Focal filling of air spaces by desquamated macrophages was also noted. There were areas of traction bronchiectasis, foci of dense chronic inflammation and peripheral bronchiolar metaplasia. Fibroblastic foci were identified as well as coalescent and mature collagen (Figure 2). Histochemical stains for fungi and mycobacteria were negative for organisms. No birefringent fibers or particles were observed. Perls’ reaction revealed positive staining outlining what appeared to be both fibers and particles (Figures 3A–3D). The iron-laden bodies varied from perfectly linear fibers, with very small core diameters, to almost particulate matter. Linear bodies were irregularly interrupted by deposition of iron-abundant material. No fiber appeared to demonstrate perfect symmetry.
Patient 2
A 41-year-old African-American female was referred with an interstitial lung disease of unclear etiology. There were complaints of dyspnea with minimal exertion and a nonproductive cough. She had smoked 2 to 1 packs of cigarettes per day for 16 years (she stopped at the time of her first being seen). Employment for 23 years had been a grader in a textile mill (different site of employment relative to patient 1); she reported no occupational exposures to asbestos, silica, or silicates. There were bibasilar crackles on examination. The FVC was 1.07 L (38% predicted), the FEV1 was 0.98 L (42% predicted), and the FEV1/FVC was 92%. The TLC was 1.98 L (41% predicted) and the diffusion capacity was not able to be measured. The chest X-ray showed diffuse interstitial markings throughout both lung fields with areas of honeycombing. The HRCT scan confirmed interstitial lung disease and honeycombing more prominent in the superior portions of the lungs. A transbronchial biopsy showed bronchiolitis with mixed lymphocytic and eosinophilic infiltration of the bronchial walls. Two courses of prednisone (each over several months) were associated with no improvement and were stopped. A thoracoscopic biopsy was obtained which demonstrated end stage fibrotic injury of the lung. No birefringent fibers or particles were observed. Iron staining revealed numerous ferruginous bodies. Positively stained bodies appeared to include both particles and fibers. These bodies varied from perfectly linear fibers, with very small core diameters, to almost particulate matter.
Lung tissue was retrieved from the paraffin block, deparaffinized in xylene, and rehydrated to 95% ethanol (wet weight of 0.281 g). This tissue was processed for digestion using the sodium hypochlorite technique (Roggli, 2004). The residue was collected on a 0.4 micron pore-size Nuclepore filter, which was then mounted on a carbon disc with colloidal graphite, sputter coated with gold, and examined by scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDXA) (Roggli et al., 1993). By SEM, there were 35,000 uncoated fibers per gram of wet lung tissue (1300 × magnification, corrected for paraffin block). Twenty consecutive fibers were examined by EDXA, 8 of which consisted of carbon and oxygen without evidence of an inorganic components (Figure 4B). Fibers varied in length, tended to be irregular in shape with knobbed or blunted ends, and had a twisted configuration (Figure 4A). EDXA also confirmed the presence of a ferruginous body associated with a nylon fiber (Figures 5A and 5B). The remaining twelve fibers included five talc, two silica, two aluminum silicates, one rutile, one iron (probably the oxide), and one sodium potassium aluminum silicate.
Results
SEM of synthetics collected at the site of employment demonstrated irregular fibers with a twisted appearance identical to those noted in the lung tissue sample digest (Figure 6A). EDXA spectra yielded a pattern comparable to that obtained from the fibers isolated from the lung tissue digest (Figure 6B). Except for the lack of birefringence, fibers collected from both the textile mill and the lung specimen were comparable in appearance to those previously isolated from individuals with Flock Worker’s Lung and rats instilled with Nylon (Boag et al., 1999).
There was a measurable concentration of iron associated with the synthetic fibers as acquired from the textile mill. This was quantified to be 0.040 μg iron/mg fiber. However, further concentrations of this metal were complexed by the synthetic fibers. It appears that the capacity of the synthetic fibers to complex iron approximated 9.20 μg/mg fiber.
Oxidant generation, as measured by TBA reactive products of deoxyribose, by fibers was significantly elevated relative to controls without the additional iron loading (Table 1). This oxidative stress presented by the synthetic fibers was increased further following complexation by iron. Both the radical scavenger DMTU and the metal chelator deferoxamine significantly diminished oxidant generation by the textile fiber measured as absorbance of TBA reactive products of deoxyribose.
Discussion
The clinical presentations of the patients were consistent with a chronic fibrotic disease of the lung with dyspnea, crackles on examination, restrictive pulmonary function, and radiographic procedures supportive of interstitial disease. While fibroblastic foci were not demonstrated, the trichrome stain showed a significant deposition of collagen. Staining of the resected tissue for iron revealed structures having the appearance of ferruginous bodies. It is proposed that these structures reflect an in vivo accumulation of metal on synthetic fibers sequestered in the lung. Alternatively, there could be additional exposures in these patients which disrupted normal iron homeostasis in the lower respiratory tract and affected an accumulation of this metal. EDXA demonstrated that fibers isolated from the lung were carbonaceous. Again, these may reflect either those structures included in the ferruginous bodies observed or a second exposure. Fibers collected from surfaces at a textile mill were remarkably similar in both appearance (using SEM) and composition (using EDXA) suggesting that the fibers sequestered in the lungs of these patients were synthetics originating from the mill.
In vitro investigation demonstrated that synthetic fibers collected from a mill had some concentration of iron comparable to both inorganic (Lund and Aust, 1991) and cellulose (Kongdee and Bechtold, 2004) fibers. There was a capacity of the synthetics to complex further amounts of iron which is also a property shared by all fibers (Lund and Aust, 1991; Kongdee and Bechtold, 2004). The composition of synthetic fibers will vary. Dacron is ethylene glycol-terephthalic acid polyester, Nylon is a polyamide, and Rayon is cellulose acetate. They have carbonyl groups (and Nylon also has -NH groups) which demonstrate a capacity to complex metals particularly iron. As a result of a lack of flexibility of a solid surface, the complexation of a metal by the surface of synthetic fibers will be incomplete with coordination sites away from the surface being either labile or vacant. This allows a participation of the metal in electron transport and catalysis of oxidants including hydroxyl radical, perferryl radical, and superoxide. Generation of TBA reactive products by the synthetic fibers was increased following exposure to iron supporting such complexation and radical production as a potential mechanism of biological effect.
Previous investigation has confirmed total dust levels as high as 40 mg/m3 in textile mills suggesting that the burden of dust in the lungs of workers in a textile mill can be considerable (Burkhart et al., 1999). This would result in some sequestration of insoluble fiber in the lower respiratory tract of textile mill workers. After complexation of metal at the surface of the fiber, an oxidative stress in the lower respiratory tract would result. This can correspond with phosphorylation-dependent cell signaling, an activation of specific transcription factors, an increased expression of mediators, and finally an inflammatory injury. This is comparable to instillation of nylon in an animal model, which resulted in an inflammatory response (Burkhart et al., 1999). The metal-catalyzed oxidative stress associated with exposure to synthetic fibers could also affect a fibrotic injury. Exposure to iron with a labile or reactive coordination site, such as the ferrous-bleomycin coordination complex, directly increases, while a metal chelator inhibits, prolyl hydroxylase activity (Hunt et al., 1979; Giri et al., 1983; Franklin et al., 1991). Finally, dietary depletion of iron and metal chelation can inhibit lung fibrosis after exposure to the iron chelate bleomycin (Chandler et al., 1988).
It is concluded that textile workers with pulmonary fibrosis can potentially demonstrate evidence of sequestered synthetic fibers with ferruginous body formation. In vitro investigation is provided to support that this accumulation of metal reflects a potential oxidative stress in the lower respiratory tract after exposure to synthetic fibers.