A comparison of cytotoxicity and oxidative stress from welding fumes generated with a new nickel-, copper-based consumable versus mild and stainless steel-based welding in RAW 264.7 mouse macrophages

Abstract

Welding processes that generate fumes containing toxic metals, such as hexavalent chromium (Cr(VI)), manganese (Mn), and nickel (Ni), have been implicated in lung injury, inflammation, and lung tumor promotion in animal models. While federal regulations have reduced permissible worker exposure limits to Cr(VI), this is not always practical considering that welders may work in confined spaces and exhaust ventilation may be ineffective. Thus, there has been a recent initiative to minimize the potentially hazardous components in welding materials by developing new consumables containing much less Cr(VI) and Mn. A new nickel (Ni) and copper (Cu)-based material (Ni-Cu WF) is being suggested as a safer alternative to stainless steel consumables; however, its adverse cellular effects have not been studied. This study compared the cytotoxic effects of the newly developed Ni-Cu WF with two well-characterized welding fumes, collected from gas metal arc welding using mild steel (GMA-MS) or stainless steel (GMA-SS) electrodes. RAW 264.7 mouse macrophages were exposed to the three welding fumes at two doses (50 µg/ml and 250 µg/ml) for up to 24 hours. Cell viability, reactive oxygen species (ROS) production, phagocytic function, and cytokine production were examined. The GMA-MS and GMA-SS samples were found to be more reactive in terms of ROS production compared to the Ni-Cu WF. However, the fumes from this new material were more cytotoxic, inducing cell death and mitochondrial dysfunction at a lower dose. Additionally, pre-treatment with Ni-Cu WF particles impaired the ability of cells to phagocytize E. coli, suggesting macrophage dysfunction. Thus, the toxic cellular responses to welding fumes are largely due to the metal composition. The results also suggest that reducing Cr(VI) and Mn in the generated fume by increasing the concentration of other metals (e.g., Ni, Cu) may not necessarily improve welder safety.

Figure 1. Welding fumes reduce RAW 264.7 cell viability.

Cells were treated with suspensions of welding fumes for 24(A) and percentage of viable cells (B) within each sample. Error bars represent the mean ± SD (n = 4). *, p<0.05 compared to PBS control. MS; GMA-MS, SS; GMA-SS, Ni; Ni-Cu WF.

Figure 2. Reduced viability is not due to excessive ROS production.

RAW 264.7 cells were pre-treated with and without N-acetyl-L-cysteine (NAC) for 1 h and then for 24 h with suspensions of welding fumes. MultiTox-Fluor Reagent (Promega) was added to all wells for 1 h at 37°C, and plates were read at 400ex/505em to determine the fluorescence signal from live cells. Error bars represent the mean ± SD (n = 3). There was no significant difference between − and + NAC for treatment groups. *, p<0.05 compared to PBS control.

Figure 3. Mitochondrial dysfunction following incubations with welding fumes.

RAW 264.7 cells were treated with suspensions of welding fumes. Following 24°C, media were removed and wells were washed 3 times with unbuffered XF media, and the plate was run using the mitochondrial stress test kit (Seahorse Biosciences) to determine the oxygen consumption rate (OCR) at certain time points. A. Representative mean OCR traces at baseline and following injections of reagents. B. ATP production, maximum respiration, and spare capacity were calculated from the mean OCRs from 5 independent experiments. Error bars represent the mean ± SD (n = 5). *, p<0.05 and **, p<0.0001 compared to PBS control.

Figure 4. Electron spin resonance measurement of •OH radicals following reaction of welding fumes with H₂O₂ or RAW 264.7 cells.

A. An acellular mixture of welding fume suspensions (1 mg/ml), H₂O₂ (1 mM), and spin trap DMPO (100 mM) in PBS were incubated in test tubes for 3 min at room temperature, and scanned via ESR. Signal intensity (peak height) is used to measure the relative amount of hydroxyl radicals and error bars represent the mean ± SD (n = 3). Inset: representative GMA-MS spectra. B. The same as in A, except without H₂O₂ but including 2×10⁶ RAW 264.7 cells/ml. Samples were incubated at 37°C for 5 min prior to measurements. (n = 5–6). Insets: representative GMA-MS spectra and with catalase treatment (+Cat) for cellular ESR. *, p<0.05.

Figure 5. Intracellular ROS production in cells following treatment with welding fumes.

A. RAW 264.7 cells were pre-treated with DCFH-DA, exposed to 50 µg/ml suspensions of welding fumes, and plates were read each hour to measure intracellular ROS. Error bars represent the mean ± SD (n = 3). *, p<0.05 compared to PBS controls at that time point. B. A comet assay was used to examine DNA damage. Cells were treated with 50 µg/ml suspensions of welding fumes for 3 h, washed and scraped into PBS, added to glass slides with agarose, then lysed and subjected to electrophoresis. SYBR green was added to stain double-stranded DNA. Images were acquired using fluorescence microscopy and a 40x objective. Experiments were performed in duplicate and comets were measured by comparing the corrected nuclear region fluorescence to the corrected total cell fluorescence. This was converted to a percentage to indicate DNA damage. Error bars represent the mean ± SD. *, p<0.05 compared to PBS controls. C. Representative images of comets.

Figure 6. Pre-incubation with Ni-Cu WF impairs phagocytosis.

RAW 264.7 cells were treated with 50 µg/ml welding fume suspensions for 3 or 6 h or cytochalasin D (Cyto D) for 3 h. Cells were washed, pHrodo Red E. coli BioParticles were added for 2 h, and plates were read to measure changes in fluorescence. Error bars represent the mean ± SD (n = 4). *, p<0.05 compared to PBS controls.

Figure 7. Welding fumes do not induce pro-inflammatory cytokine production.

RAW 264.7 cells were treated with welding fume suspensions or 1 µg/ml LPS for 24 h. Media were collected, and ELISAs were run to measure levels of TNFα (A), IL-6 (B) and IL-1β (C). Error bars represent the mean ± SD (n = 3). *, p<0.05 compared to PBS controls.