* Center for Occupational Toxicology, Occupational Safety and Health Research Institute, Korea Occupational Safety and Health Agency, 1048 Moonji-dong, Yusung-gu, Taejon, 305380, Korea;
College of Pharmacy, Sung Kyun Kwan University, Suwon, 440-74 Korea;
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
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ABSTRACT |
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Key Words: welding fumes; stainless steel; fibrosis; lung; manual metal arc welding; inhalation; rats.
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INTRODUCTION |
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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., 1983; Kalliomaki et al., 1986
; Uemitsu et al., 1984
), 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., 2000
). 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.
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MATERIALS AND METHODS |
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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 5676 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 105118 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, 1990).
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RESULTS |
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Organ Weights
As shown in Figure 2A, 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. 2B
). 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. 2B
). 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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DISCUSSION |
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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., 1986; Kurokawa et al., 1998
).
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., 1997) and in vivo studies using the instillation method (Antonini et al., 1997
; Kalliomaki et al., 1986
; Toya et al., 1999
), and in vivo studies using the inhalation method (Hicks et al., 1983
; Uemitsu et al., 1984
). 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., 2000). 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., 2000
), 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., 2000). 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., 2000). 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.
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NOTES |
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