Soluble transition metals cause the pro-inflammatory effects of welding fumes in vitro

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Abstract

Epidemiological studies have consistently reported a higher incidence of respiratory illnesses such as bronchitis, metal fume fever (MFF), and chronic pneumonitis among welders exposed to high concentrations of metal-enriched welding fumes. Here, we studied the molecular toxicology of three different metal-rich welding fumes: NIMROD 182, NIMROD c276, and COBSTEL 6. Fume toxicity in vitro was determined by exposing human type II alveolar epithelial cell line (A549) to whole welding fume, a soluble extract of fume or the “washed” particulate. All whole fumes were significantly toxic to A549 cells at doses >63 μg ml−1 (TD 50; 42, 25, and 12 μg ml−1, respectively). NIMROD c276 and COBSTEL 6 fumes increased levels of IL-8 mRNA and protein at 6 h and protein at 24 h, as did the soluble fraction alone, whereas metal chelation of the soluble fraction using chelex beads attenuated the effect. The soluble fraction of all three fumes caused a rapid depletion in intracellular glutathione following 2-h exposure with a rebound increase by 24 h. In addition, both nickel based fumes, NIMROD 182 and NIMROD c276, induced significant reactive oxygen species (ROS) production in A549 cells after 2 h as determined by DCFH fluorescence. ICP analysis confirmed that transition metal concentrations were similar in the whole and soluble fractions of each fume (dominated by Cr), but significantly less in both the washed particles and chelated fractions. These results support the hypothesis that the enhanced pro-inflammatory responses of welding fume particulates are mediated by soluble transition metal components via an oxidative stress mechanism.

Introduction

At present, it is estimated that more than one million workers are employed as welders worldwide (Sundin, 1998), with more than three million performing welding intermittently as part of their work duties (Sferlazza and Beckett, 1991). Manual metal arc welding (MMAW) and flux core arc welding (FCAW) are two commonly used procedures in the construction industry and in numerous other industrial processes. Welding involves the fusion of metals by high temperature generated via an electrical arc resulting in the formation of metal-enriched fumes. Welding fumes are a complex mixture of gases and small particulates of metal oxides formed by the vaporization and oxidation of metal during the welding process Lockey et al., 1988, Yu et al., 2000. The nature of respirable fumes depends upon the type of welding and the composition of electrode, filler wire, and fluxes Antonini et al., 1996, Lockey et al., 1988, Sferlazza and Beckett, 1991, Yu et al., 2000.

Numerous studies have reported welding fume particles to be well within the respirable size range <1 μm (reviewed by Antonini et al., 1998, Lockey et al., 1988, Yu et al., 2000). Thus, upon inhalation, particle deposition occurs in the lower respiratory tract, including the terminal bronchioles and alveoli, beyond the mucociliary escalator Sferlazza and Beckett, 1991, Yu et al., 2000.

The health of welders has been studied extensively Doig and Duguid, 1951, Sferlazza and Beckett, 1991. A number of epidemiological studies have reported a higher incidence of respiratory illness such as bronchitis, airway irritation, metal fume fever (MFF), chemical pneumonitis, and also changes in lung function in welders Antonini et al., 1996, Sferlazza and Beckett, 1991. Furthermore, the severity, duration, and frequency of acute upper and lower respiratory tract infections have been shown to be greater among welders compared to the general population (Howden et al., 1988).

The most frequently described respiratory illness among welders is metal fume fever (MFF), which is an acute, self-limiting, systemic, febrile illness caused by inhalation of high concentrations of metal oxides, primarily zinc oxide Blanc et al., 1993, Sferlazza and Beckett, 1991. Symptoms include high fever and sweating, throat irritation, chest tightness, dry cough, and general malaise associated with a pulmonary inflammatory cellular response Kuschner et al., 1995, Lockey et al., 1988, peaking 5–12 h postexposure and resolving within 24–48 h Antonini et al., 1996, Sferlazza and Beckett, 1991. Although the pathogenesis of MFF is poorly understood, allergic and immunological mechanisms are most frequently postulated (Graeme and Pollack, 1998). Cytokine networking mediated by the release of pro-inflammatory cytokines TNF-α and IL-8 by pulmonary macrophage causes both local pulmonary inflammatory cellular response and systemic response Blanc et al., 1993, Gordon, 1991, Kuschner et al., 1995. This theory is consistent with evidence showing that tolerance to metal fumes develops and symptoms are not observed on successive days of fume exposure Blount, 1990, Graeme and Pollack, 1998, Nemery, 1990.

Reports of a dose-dependent increase in pro-inflammatory cytokines and neutrophilia in the human bronchoalveolar lavage (BAL) 20–22 h following zinc oxide inhalation provide further evidence supporting the role of a cytokine-mediated mechanism causing the symptoms of MFF Blanc et al., 1993, Kuschner et al., 1995. Furthermore, TNF-α levels were significantly greater 3 h postexposure, suggesting an initial role for TNF-α in the pathophysiology of MFF Blanc et al., 1993, Kuschner et al., 1997. Similar finding were reported in rats following intratracheal installation of stainless steel welding fumes with increased levels of TNF-α and IL-β in BALF (Antonini et al., 1996). These cytokines, produced predominantly by alveolar macrophages, are involved in numerous inflammatory processes such as neutrophil recruitment and increased oxygen radical production Driscoll et al., 1990, Driscoll et al., 1991, Goldring and Krane, 1986, Schmidt et al., 1982, Tsujimoto et al., 1986. These findings provide further evidence supporting the role of cytokines in the inflammatory response associated with exposure to certain environmental and occupational particles (Antonini et al., 1996).

The importance of metal content, metal bioavailability, and interactions among transition metals in mediating pulmonary inflammation and injury following exposure to many different particles has been demonstrated. Such particles include residual oil fly ash (ROFA), stone quarry particles (Hetland et al., 2001), Provo PM10 (Costa and Dreher, 1997), and ambient airborne particles Costa and Dreher, 1997, Dreher et al., 1997, Gilmour et al., 1996. Furthermore, the pathogenicity associated with exposure to crocidolite-asbestos has been linked to the high iron content on fiber surfaces Jiménez et al., 2000, Weitzman and Weitberg, 1985.

Mechanistic studies report that particle-associated transition metals can undergo redox cycling resulting in the production and release of reactive oxygen species (ROS). These reactive compounds can deplete antioxidants, cause cellular damage, lung injury, and inflammation Carter et al., 1997, Dreher et al., 1997, Jiménez et al., 2000, Stohs and Bagchi, 1995, Toyokuni, 1996.

The aims of this study were to investigate the molecular toxicology of three compositionally different welding fumes by comparing their potential to activate lung epithelial cells to release the pro-inflammatory cytokine IL-8. We hypothesized that transition metals present in or on welding fume particles cause pulmonary inflammation and lung injury observed in welders. In addition, we postulated that the soluble metal components were the main contributing factors.

To investigate this we initially performed experiments to determine the toxicity of three different welding fumes on alveolar epithelial cells. To examine the possible mechanisms by which nontoxic concentrations of welding fume particles may cause lung inflammation, the ability of fumes to cause expression of the pro-inflammatory cytokine IL-8 in epithelial cells was assessed. A recent study by Antonini et al., 1999 identified the soluble components of fume from stainless steel MMAW to be most cytotoxic and have the greatest effect on macrophage function. Similar studies using other environmental particulates have shown that the soluble metal fraction plays an important role in the induction of lung injury Dreher et al., 1997, Adamson et al., 1999. Therefore, to determine which components of welding fume particles were responsible for the pro-inflammatory response, alveolar epithelial cells were exposed to soluble, insoluble, or whole fume components at subtoxic concentrations. In addition, we investigated whether welding fumes exerted some of their effects via oxidative stress by assessing the production of intracellular ROS and levels of the important antioxidant glutathione.

Section snippets

Materials

All reagents were obtained from Sigma-Aldrich, UK unless otherwise stated.

Cell culture

The type II human alveolar-like epithelial cell line A549 (European Collection for Animal Cell Culture) was maintained in continuous culture in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamate, and 100 IU ml penicillin−1·100 μg ml−1 streptomycin−1. Cells were grown to confluency at 37 °C in a humidified atmosphere containing 5% CO2, washed with Ca2+

Welding fume characterization

TEM images of welding fume particles are shown in Fig. 1. All welding fumes were heterogeneous in nature containing a high proportion of ultrafine (uf) particles with some larger particles. Images of carbon black and ultrafine carbon black (Figs. 1D and E) are shown for comparison.

Cytotoxicity assessments

To establish the potential toxicity of the three welding fumes NIMROD 182, NIMROD c276, and COBSTEL 6, alveolar epithelial cells were incubated with welding fumes (1–250 μg ml−1) for 24 h and LDH release was measured

Discussion

The purpose of this study was to investigate the specific role of particle-associated transition metals in welding fumes in initiating pro-inflammatory effects in alveolar epithelial cells in vitro, to determine the importance of soluble metal in the welding fumes' ability to cause inflammation.

All of the fumes were cytotoxic at high doses (>63 μg ml−1); however, following inhalation exposure, the deposited mass is likely to be very low due to effective clearance mechanisms such as the

Conclusion

In conclusion, these studies demonstrate that the soluble fractions of welding fumes play a fundamental role in mediating pro-inflammatory responses in alveolar epithelial cells as shown by increased expression of IL-8. Further examination highlighted that the soluble metal component was entirely responsible for this effect. In addition, enhanced levels of ROS and concomitant depletion of the antioxidant GSH suggest an important role for metal-particulate mediated oxidative stress, although

Acknowledgements

JMcN is the recipient of a Colt Foundation Fellowship in Occupational/Environmental Health.

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