Lung Fibrosis in Sprague-Dawley Rats, Induced by Exposure to Manual Metal Arc–Stainless Steel Welding Fumes

Il Je Yu*,{dagger},1, Kyung Seuk Song*, Hee Kyung Chang{ddagger}, Jeong Hee Han*, Kwang Jin Kim*, Yong Hyun Chung*, Seung Hee Maeng*, Seung Hyun Park*, Kuy Tae Han§, Kyu Hyuk Chung{dagger} and Ho Keun Chung*

* Center for Occupational Toxicology, Occupational Safety and Health Research Institute, Korea Occupational Safety and Health Agency, 104–8 Moonji-dong, Yusung-gu, Taejon, 305–380, Korea; {dagger} College of Pharmacy, Sung Kyun Kwan University, Suwon, 440-74 Korea; {ddagger} Department of Pathology, College of Medicine, Kosin University, Puson, 602-702 Korea; and § Korea Dusturbo Incorporation, Seoul, 152-082 Korea

Received December 21, 2000; accepted May 22, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the disease process of pneumoconiosis induced by welding-fume exposure, a lung fibrosis model was established by building a stainless steel arc welding fume generation system and exposing male Sprague-Dawley rats for 90 days. The rats were exposed to welding fumes with concentrations of 57–67 mg/m3 (low dose) and 105–118 mg/m3 (high dose) total suspended particulates for 2 h per day in an inhalation chamber for 90 days. The concentrations of the main metals, Fe, Mn, Cr, and Ni, were measured in the welding fumes, plus the gaseous compounds, including nitrous gases and ozone, were monitored. During the exposure period, the animals were sacrificed after the initial 2-h exposure and after 15, 30, 60, and 90 days. Histopathological examinations were conducted on the animals' upper respiratory tract, including the nasal pathway and conducting airway, plus the gas exchange region, including the alveolar ducts, alveolar sacs, and alveoli. When compared to the control group, the lung weights did not increase significantly in the low-dose group, yet in the high-dose group there was a significant increase from day 15 to day 90. The histopatholgical examination combined with fibrosis-specific staining (Masson's trichrome) indicated that the lungs in the low-dose group did not exhibit any progressive fibrotic changes. Whereas, the lungs in the high-dose group exhibited early delicate fibrosis from day 15, which progressed into the perivascular and peribronchiolar regions by day 30. Interstitial fibrosis appeared at day 60 and became prominent by day 90, along with the additional appearance of pleural fibrosis. Accordingly, it would appear that a significant dose of welding-fume exposure was required to induce lung fibrosis.

Key Words: welding fumes; stainless steel; fibrosis; lung; manual metal arc welding; inhalation; rats.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chest x-rays of welders indicate an increased profusion of small opacities due to the chronic inhalation of welding fumes. Exposure to iron oxide fumes would appear to cause iron oxide pneumoconiosis (called siderosis), a prominently abnormal chest film with no impairment of pulmonary function, attributable to welding-fume exposure (Beckett, 1996Go). Welders with siderosis have shown a gradual clearing of the x-ray identified effects following removal from exposure. In a 1978 survey of electric arc welders in Britain, 7% reported some degree of pneumoconiosis, yet none showed progressive massive fibrosis (Artfield and Ross, 1978Go). In contrast, functionally significant pulmonary fibrosis associated with exposure to silica dust, nitrogen dioxide gas, and other components of the welding fume would appear to lead to a more severe form of fibrosis in welders (Funahashi et al., 1988Go; Guidotti et al., 1978Go). Welding-fume exposure–related pneumoconiosis is still prevalent in some countries (Lubianova, 1990Go; Nemery, 1990Go; Ruegger, 1995Go; Steurich and Feyerabend, 1997Go). In particular, welders working in a confined space, as in the shipbuilding industry, have a higher risk of exposure to high concentrations of welding fumes and of developing pneumoconiosis. A previous study indicated that the annual prevalence rate of welders' pneumoconiosis (profusion 0/1 or above) from a shipyard in Pusan, Korea was 8.9% (92/1062) in 1986 and 7.9% (43/504) in 1993. The radiographic abnormalities of the pneumoconiosis partially or completely disappear when welders are removed from fume exposure (Sohn et al., 1994Go). In vitro studies indicate that hexavalent chromium from stainless steel welding and nitrogen dioxide are candidate causative substances (Stern et al., 1983Go). The most common type of welding, manual metal arc welding (MMA), combined with stainless steel (MMA-SS) is known to be associated with higher emissions of toxic compounds (Kalliomaki et al., 1986Go). MMA-SS welding is also known to be more cytotoxic to macrophages and induce a greater release of reactive oxygen species than the fumes from other types of welding (Antonini et al., 1997Go; 1999).

Several studies on inhalation or instillation exposure have been conducted to induce lung fibrosis based on the repeated exposure of animals to welding fumes (Hicks et al., 1983Go; Kalliomaki et al., 1986Go; Uemitsu et al., 1984Go), however, the disease process of lung fibrosis induced by welding-fume exposure has not yet been completely elucidated. In a previous study by the current authors, a novel welding fume-generating system was developed for the long-term exposure of experimental animals (Yu et al., 2000Go). Accordingly, to study the pathological process of lung fibrosis induced by welding-fume exposure, a lung fibrosis model was developed. After exposing rats to MMA-SS welding for 90 days, interstitial lung fibrosis was induced.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Generation of MMA-SS welding fumes.
The welding fumes were generated as described in (Yu et al., 2000Go); that is, the fumes were generated using a rotating stainless steel disc plate (SUS 304, 50 mm diameter, 1 cm thick) as the base metal, and a welding rod (KST 308, 26 x 300 mm, Korea Welding Electrode Co. LTD, Seoul, Korea) was restrained in a welding-rod holder support (Fig. 1AGo). When the welding rod was moved by a pulley and approached the rotating disc, an arc was produced and the rod consumed, thereby generating welding fumes. The fumes then moved into an exposure chamber (whole body type, 1.3 m3, Dusturbo, Seoul, Korea; Fig. 1BGo).



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FIG. 1. (A) Welding rod with rotating stainless steel disc. (B) Welding fume generation system.

 
Measurement of diameters of welding fume particles.
An Anderson sampler (AN-200, Shibata, Tokyo, Japan) was used to measure the mass median aerodynamic diameters of the welding fumes. The flow rate was 28.3 liters/min and the sampling time 5 min.

Analysis of welding fumes.
The welding fumes in the chamber were sampled using a personal sampler (MSA 484107, Pittsburgh, PA) with a flow rate of 2 liter/min. The welding fume particulate captured on the membrane filters (pore size 0.8 µm, 37 mm diameter, Millipore AAWP 03700, Bedford, MA) was analyzed for its metal composition with an inductively coupled plasma analyzer (Thermojeralash, IRIS, Houston, TX) using NIOSH 7300 method (1999). NIOSH method 7604 (1999) was used to evaluate the CrVI in the welding fumes. The samples used for measuring the total Cr were stored in vials containing a base solution (2% NaOH, 2% Na2CO3), whereas the samples for measuring CrVI were kept in vials containing deionzed water. The total Cr concentration was measured using an atomic absorption spectrophotometer (SpectAA-800, Varian, Palo Alto, CA) after a pretreatment of ashing with a microwave oven. The CrVI concentration was measured using an ion chromatography (DX-500, Dionex, Sunnyvale, CA) after extracting the base and deionized water.

The gaseous fumes, O3, NO2, and nitrous fumes, were measured using Dräger tubes (Cat No. 6733181, CH 31001, and CH 30001, respectively). The gaseous fumes were sampled by stroking a gas detector pump (6400000, Dräger, Lübeck, Germany), following the manufacturer's directions, 1 h after the welding-fume exposure began.

Study of inhalation toxicity.
Two 90-day inhalation toxicity studies were conducted. In the first study, 6-week-old male, specific pathogen-free (SPF) Sprague-Dawley rats, purchased from the Daehan Center (Korea), were acclimated to a 12-h light/dark cycle with light from 0800 to 2000 h. The rats were fed Purina Chow 5001 (Ralston Purina Co., St Louis, MO) and tap water ad libitum for 1 week before the initiation of the experiment. Rats weighing 194 ± 7 g were randomly assigned to 6 groups and exposed to welding fumes for 2 h per day in the exposure chamber. One group was sacrificed immediately after the first 2-h exposure, while the other groups were sacrificed after 15, 30, 60, or 90 days of exposure. Each group consisted of 5 unexposed and 5 exposed rats. Three rats from each group were used for histopathological examination and 2 rats were processed to determine the organ distribution of the metal components from the welding fumes. The TWA concentration of the exposure ranged from 56–76 mg/m3 per 2 h (low dose).

In the second study, 5-week-old male SPF Sprague-Dawley rats, purchased from the Charles River Laboratory (Japan) and weighing 134 ± 7 g, were randomly assigned to 6 groups and exposed to welding fumes for 2 h per day in the exposure chamber. One group was sacrificed immediately after the first 2-h exposure, while the other groups were sacrificed after 15, 30, 60, or 90 days of exposure. Each group consisted of 4 exposed and 4 unexposed rats. The TWA concentration of the exposure ranged from 105–118 mg/m3 per 2h (high dose).

Histopathology.
After exposure, the rats were anesthetized with diethyl ether, and blood was collected from the abdominal aorta. The lungs, trachea, and other organs including the adrenals, testes, heart, kidneys, spleen, liver, and brain, were removed and fixed in a 10% formalin solution containing neutral phosphate buffered saline. Histological specimens from the nasal pathways were prepared using the method described by Yu et al. (2000). The nasal pathways were gently flushed via the nasopharyngeal duct with 10% neutral phosphate buffered formalin (NPBF). The samples were then fixed in 10% NPBF for 7 days and delipidized by incubation with 70% methanol for 1 h, methanol-chloroform (1:1) for 1 h, and 70% methanol for 1 h. The samples were decalcified with 5% formic acid for at least 7 days and stored in NPBF until processing. Three histological sections were made from the proximal nasal pathway to the distal nasal pathway: incisor teeth, incisive papilla, and molar teeth region. The specimens were embedded in paraffin, stained with hematoxylin and eosin (H & E), and examined by light microscopy. Some specimens were stained with Masson's trichrome to visualize any fibrosis (Carson, 1990Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MMA-SS Welding Fume Characterization
The aerodynamic diameters of the welding fume particles are shown in Table 1Go. More than 90% of the fume particles had diameters of less than 1 µm. Fifty percent of the diameters were between 0.65 and 0.43 µm. The MMA-SS welding fume consisted of mainly Fe, Mn, Cr, and Ni. The metal concentrations and gaseous fractions of the welding fumes are shown in Table 2Go.


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TABLE 1 Size Distribution of Stainless Steel Welding Fume Particles Using Anderson Sampler
 

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TABLE 2 Concentrations of MMA-SS Welding Fume Components
 
Body Weight Development and Animal Observation
The rats exposed to the welding fumes showed no statistically significant changes in body weight during the 90-day experiment. In addition, the welding fume-exposed animals did not show any distinct behavioral changes.

Organ Weights
As shown in Figure 2AGo, the lung weight of the rats exposed to a low dose of welding fumes did not show any significant weight increase compared to the lung weight of the control during the 90-day exposure period. In contrast, the lung weight of the rats exposed to a high dose of welding fumes showed a significant (p < 0.01) weight increase compared to the control (Fig. 2BGo). The lung weight increase was initially evident after 15 days of exposure and continued to increase until day 90. Compared to the control, the lung weight increase of the exposed group showed an initial sharp increase up to 30 days, followed by a stable period between 30 and 60 days. Another sharp increase was noted between 60 and 90 days of exposure (Fig. 2BGo). No other organ weights, including the adrenals, testes, kidneys, heart, spleen, liver, and brain showed any significant increase at any of the necropsy points (after 2 h, and after 15, 30, 60, and 90 days).



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FIG. 2. Lung weight changes of rats during 90 days of MMA-SS welding-fume exposure. The lung weights were measured after the initial 2 h exposure and after 15, 30, 60, and 90 days of exposure. (A) Male rats exposed to low dose (56–76 mg/m3). (B) Male rats exposed to high dose (105–118 mg/m3).

 
Gross and Histopathological Examination
The pleural surfaces of the rats exposed to high doses appeared white to grayish, and round elevated foci were evident on the lung surfaces, with atrophy (data not shown). Initially, this phenomenon was considered to be a minor effect, and a tendency towards confluence was observed. The incidences and types of fibrosis observed after 90 days of welding-fume exposure are summarized in Table 3Go. The low-dose group did not show any progressive fibrotic regions (Fig. 3Go). Although the focal accumulation and mobilization of lymphocytes and particle-laden macrophages to the interstitial and alveolar regions were evident from day 15–60 (Figs. 3BGo, arrow and 3C, open triangle), only early delicate fibrosis or equivocal fibrosis was detected around the particle-laden macrophages when using trichrome staining to visualize the fibrosis (Fig. 3DGo, filled triangle). The histopathological examinations of the lung samples from the high-dose group after the initial 2 h welding-fume exposure showed particle-laden macrophages in the small bronchioles (Fig. 4AGo, filled triangles), yet no distinct fibrosis in the bronchioles or lung parenchyma (Fig. 4CGo). After 15 days of high-dose welding-fume exposure, the histopathological evaluation of the lungs indicated perivasculitis (Fig. 4BGo, filled square) and delicate early fibrosis around the peribronchioles and perivascular regions (Fig. 4DGo, arrows) when using trichrome staining to visualize the fibrosis. After 30 days of high-dose welding-fume exposure the lungs showed particle-laden macrophages in the alveolar spaces (Fig. 5AGo, filled triangles) and early fibrosis around the perivascular areas (Fig. 5CGo, arrows). The particle-laden macrophages in the bronchioles were indicative of alveolar lipoproteinosis or multifocal histiocytosis (19). After 60 days of high-dose welding-fume exposure, the lungs showed granulomatous regions and distinct fibrosis around the perivascular and peribronchiole areas (Fig. 5BGo, stars) plus some interstitial fibrosis (Fig. 5DGo, arrows). After 90 days of high-dose welding-fume exposure, welding fume particle-loaded macrophages filled the alveolar spaces, and some macrophages had accumulated many particles and formed macules (Fig. 6AGo, triangles). In addition, many granulomatous regions were observed (Fig. 6BGo, stars) and interstitial fibrosis and peripleural fibrosis were noted (Fig. 6CGo, arrows). The histopatholgical examination of the nasal pathway, including the squamous, transitional, respiratory, and olfactory epithelium, did not reveal any significant damage to the cells in the nasal pathway regions. Neither the trachea nor the large bronchi showed any significant histopathological changes or adsorption of fume particles.


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TABLE 3 Types of Fibrosis Observed after 90 Days of Welding-Fume Exposure
 


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FIG. 3. Histopathology of lungs of rats exposed to low dose of MMM-SS welding fumes for 90 days. (A) Control, (B) after 30 days of exposure, (C) after 60 days of exposure, (D) after 90 days of exposure. Samples were stained with trichrome. The arrow indicates the accumulation and mobilization of lymphocytes and macrophages in the interstitium and alveolar region. The open triangle indicates the focal accumulation of lymphocytes around the bronchus and bronchioles. The filled triangle indicates the nodular aggregation of foamy cells in which equivocal delicate fibrosis (bluish color) can be seen. Scale bar is 100 µm.

 


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FIG. 4. Histopathology of lungs of rats exposed to MMM-SS welding fumes for 2 h and 15 days. (A & C) After initial 2 exposure, (B & D) after 15 days of exposure, (A & B) H & E staining, (C & D) trichrome staining. The filled triangles indicate welding fume-ingested alveolar macrophages. The filled square indicates perivasculitis. The arrows indicate fibrosis stained by trichrome staining (bluish color). Scale bar is 100 µm.

 


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FIG. 5. Histopathology of lungs of rats exposed to MMM-SS welding fumes for 30 and 60 days. (A & C) After 30 days of exposure, (B & D) after 60 days of exposure, (A & B) H & E staining, (C & D) trichrome staining. The filled triangles indicate welding fume-ingested alveolar macrophages. The arrows indicate fibrosis in the interstitia and peribronchiolar and perivascular areas stained by trichrome staining (bluish color). The stars indicate granulomatous regions. Scale bar is 100 µm.

 


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FIG. 6. Histopathology of lungs of rats exposed to MMM-SS welding fumes for 90 days. (A) Particle-laden macrophages and macules in alveolar regions (filled triangles). (B) Granulomatous regions and fibrotic regions. (C) Interstitial fibrosis (arrows) and perivascular fibrosis (X). (A & B) H&E staining, (C) trichrome staining. Scale bar is 100 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously, it was demonstrated that welding fume particles are mainly adsorbed in the lower respiratory tracts, including the bronchioles, alveolar ducts, alveolar sacs, and alveoli when rats are exposed short-term in an inhalation system (Yu et al., 2000Go). Using the same inhalation for long-term exposure, the present study demonstrated that continuous exposure to MMA-SS welding fumes induced a dose-dependent interstitial fibrosis 60 days after the initial exposure. The selection of doses in the current study may appear to be too high to evaluate lung fibrosis; however, the doses were selected based on actual exposure monitoring data. Several domestic monitoring studies have reported that welding-fume concentrations can be higher than TLV 5 mg/m3 and lower than the high dose used in the current study. Several studies on the monitoring of exposure to welding fumes in the shipbuilding industry have reported on concentrations ranging from 6–73 mg/m3 (geometric mean [GM] 16.6 mg/m3) to 0.3–91.16 mg/m3 (GM 5.59 mg/m3; Choi et al., 1999; Kwag and Paik, 1997). In addition, the current exposure duration was only 2 h compared with 6 h in most inhalation studies or 8 h in actual workplace monitoring. In the present study it was presumed that the actual exposure to welding fumes in the workplace could be 20–30 mg/m3 for longer than 10 years in a severe case. Thus, in terms of a 6-h exposure, a low and high dose would be 20 and 40 mg/m3, respectively. Although the exact causative agents for lung fibrosis are not clear from the current experiment, and both the particulate fraction and the gaseous fraction may contribute to the induction of fibrosis, the dose response of welding-fume exposure clearly indicated that the particulate fraction would appear to play a role in the induction of fibrosis. The current results suggest that if workers are exposed long-term to welding fumes at high concentrations several fold over TLV in a confined space, as in the shipbuilding industry or other occupational settings, lung fibrosis will develop. However, if workers are exposed long-term to lower concentrations around TLV, they will not develop lung fibrosis.

Since welding fumes have a very small average diameter and heterogeneous composition, it is difficult to select a proper control particle for fume exposure experiments. Furthermore, it is really difficult to separate the particulate fractions from the gaseous fractions in welding fumes to establish the causal relationship of fibrosis, as the particulate usually contains gaseous fractions concurrently (Knecht et al., 1986Go; Kurokawa et al., 1998Go).

In the current model, lung fibrosis induced by welding-fume exposure appeared to have 3 phases of particle accumulation based on comparing the fume-exposed lung weight to the control. The first phase, up to 30 days after the initial exposure, was the initial overloading phase, characterized by a rapid increase in the lung weight. During this phase, perivasculitis with fibrosis began to appear in the perivascular and peribronchiolar regions. The recruitment of macrophages to the alveolar airspace was expected to be the main event during this phase; however, the macrophages may not have reached their maximum capability to clear off the deposited fume particles. The second phase from day 30 to day 60, was the steady phase, characterized by a stagnancy in the lung weight increase and an equilibrium between the overloading and the clearance of the welding fume particles in the lung. Granulomatous regions and distinct fibrosis around the perivascular and peribronchiole areas and some interstitial fibrosis were also evident during this period. The recruitment of macrophages was in full force and the clearance capability of the macrophages was maximized. Some macrophages burst after ingesting and accumulating fume particles in their cytoplasm, as a result, macular shapes of macrophage-accumulated fume particles appeared in the alveolar space as well as in the interstitial regions. At this point the fibrosis could not be stopped even after the exposure was stopped, as such, this effect was irreversible. The third phase, from day 60 to day 90, was the second overloading phase, characterized by a sharp increase in the lung weight, interstitial fibrosis, and pleural fibrosis. The lung lost its capability to clean up the accumulated fume particles, and the recruitment of macrophages could not overcome the lung overload of fume particles. The fibrosis could not be stopped even after the exposure was stopped. Accordingly, the current lung fibrosis model, induced by welding-fume exposure, clearly described triphasic steps for lung fibrosis that have not been observed in other studies, including in vitro (Antonini et al., 1997Go) and in vivo studies using the instillation method (Antonini et al., 1997Go; Kalliomaki et al., 1986Go; Toya et al., 1999Go), and in vivo studies using the inhalation method (Hicks et al., 1983Go; Uemitsu et al., 1984Go). The current study maintained consistent fume concentrations and simulated actual workplace exposure (confined space).

When analyzed by an electron microscope, the particles were log-normally distributed with diameters ranging from 0.023 to 0.81 µm, with a 0.1 µm geometric mean diameter (Yu et al., 2000Go). The 5 or 6 discrepancies compared to the Anderson sampler measurements may have been due to the density of the fume particles. As seen with short-term exposure (Yu et al., 2000Go), the welding fume particles were too small to be adsorbed in the nasal pathways and conducting airways in the current 90-day exposure experiment. The effect of particles and gases on the nasal pathways and conducting airways is currently under investigation.

The gaseous fraction of the welding fume, CrVI, ozone, nitrogen dioxide, and nitrous fumes, could have additively or synergistically contributed to the lung fibrosis. Exposure to 0.4 ppm ozone and 7 ppm nitrogen dioxide has induced pulmonary fibrosis in Sprague-Dawely rats after 60 to 90 days of exposure (Ishii et al., 2000Go). Although the ozone and nitrogen dioxide concentrations were lower in the current study (0.1 and 0.5 ppm, respectively), the exposure to these gases could have also stimulated the particle exposure-induced lung fibrosis.

The activation of macrophages by ingesting fume particles or by exposure to gaseous fractions of welding fumes can produce fibrogenic cytokines without the persistent inflammation of neutrophils (Ishii et al., 2000Go). In the current experiment, the histopathologic evaluation of the lung sections did not show any significant neutrophil response, yet indicated macrophage mobilization and response from the initial exposure to day 90. Thus, the fibrosis caused by the MMA-SS welding fumes in the current study appeared to originate from factors released from the welding fume-exposed macrophages.


    NOTES
 
1 To whom correspondence should be addressed. Fax: 82–42–863–8361. E-mail: u1670916{at}chollian.net. Back


    REFERENCES
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