Chelex 100 resin was purchased from Bio-Rad Laboratories (Richmond, CA, USA). Phosphate-buffered saline (PBS), (KH2PO4 (1.06 mM), Na2HPO4 (5.6 mM), NaCl (154 mM), pH 7.4), was purchased from Biowhittaker Inc. (Walkersville, MD, USA). The PBS was treated with Chelex 100 to remove transition metal ion contaminants. Dulbecco's modified eagles medium (DMEM), 5,5-dimethyl-1-pyroline-oxide (DMPO), fetal bovine serum (FBS), FeSO4, H2O2, and penicillin/streptomycin were purchased from Sigma Chemical Company (St. Louis, MO, USA). The spin trap, DMPO, was purified by charcoal decolorization and vacuum distillation and was free of ESR detectable impurities. Quartz sample tubes were purchased from Wilmad Glass (Buena, NJ, USA).
RAW 264.7 mouse peritoneal monocytes were purchased from American Type Culture Collection (Rockville, MD). RAW 264.7 cells are commonly used and have been found to respond to particle exposure in a manner similar to primary alveolar macrophages [46
]. RAW 264.7 cells were cultured in DMEM with 10% FBS, 2 mM L-glutamine, and 50 mg/ml pen/strep at 37°C in a 5% CO2
incubator. Cells were split after confluence approximately every 3 days.
Generation and collection of welding fumes
The welding fume generation system was similar to that previously outlined [22
]. Briefly, the generation system consisted of a welding power source (Power Wave 455, Lincoln Electric, Cleveland, OH), an automated, programmable six-axis robotic arm (Model 100 Bi, Lincoln Electric), a water cooled arc welding torch (WC 650 amp, Lincoln Electric), a wire feeder that supplied the wire to the torch at a programmed rate up to 300 inches/min, and an automatic welding torch cleaner that kept the welding nozzle free of debris and spatter. Gas metal arc welding was performed using a mild steel electrode (carbon steel ER70S-6, Lincoln Electric) or a stainless steel electrode (Blue Max E308LSi wire, Lincoln Electric). Welding took place on A36 carbon steel plates for fume collection times of 20 min at 25 V and 200 amps. During welding, a shielding gas combination of 95% Ar and 5% CO2
(Airgas Co., Morgantown, WV) was continually delivered to the welding nozzle at an air flow of 20 L/min.
Figure shows an outline of the generation and collection system. The system is contained in three rooms; the control room, the robotic arm welding fume generator and the fume collection chamber. All three rooms were separated from each other.
Aerodynamically size-selected aerosol samples were collected with the Micro-Orifice Uniform Deposit Impactor (MOUDI), model # 110 and Nano-MOUDI MSP Model #115, with rotator (MSP, Inc., Minneapolis, MN, USA). This MOUDI/Nano-MOUDI arraignment provided a 15-stage research-grade cascade impactor. Each filter stage has a cutoff size with particles collected at each stage being aerodynamically size-selected between stage sizes. Cutoff sizes were; 18, 10, 5.6,3.2, 1.8, 1.0, 0.56, 0.32, 0.18, 0.10, 0.006, 0.03, 0.02, 0.01 μm and the final filter. Filters used for ESR and cellular exposures were from Millipore Corp. (Billerica, MA, USA) 47 mm, 0.8 μm, PVC model PVC0847600. PVC was selected because it has previously been demonstrated to have no effect in the free radical analysis. The MOUDI substrates are normally coated with grease to ensure adherence of deposited particles and to avoid bounce of large particles to lower stages of the impactor. However, grease can alter the surface of collected aerosol particles and is not suitable for use in collecting samples for free radical analysis. Therefore, the cascade impactor was operated without grease substrates to collect and fractionate the welding fume. Filters were collected for 20 min. Filters were then split into 3 groups for either 1 hour, 24 hour or 1 week post generation analysis.
Filter suspensions, which were used for H2O2, lipid peroxidation, oxygen consumption and DNA damage analysis, were prepared by splitting the filters into three size groups. For our study the size groups were defined as ultrafine (0.01 - 0.056 μm), fine (0.1 - 1.0 μm) and coarse (1.8 - 18 μm) particles. The groups consisted of five filters each of which were placed in PBS. Pre- and post filter weights were used to prepare a final concentration of 1 mg/ml welding fume suspension. The slurry was centrifuged, and the fume suspension was decanted from the filter pellet. A clean control suspension was also prepared at a ratio of 4 filters/1 ml PBS.
Welding particle morphology, transmission electron microscopy (TEM)
Welding fume samples, collected separately from the fumes used in free radial analysis, were collected at 30-minute intervals during 3 h of welding directly onto formvar-coated TEM grids and viewed using a JEOL 1220 transmission electron microscope (JOEL, Inc.).
Welding particle size distribution
Particle size distribution was determined by using a Micro-Orifice Uniform Deposit Impactor (MOUDI) model # 110 which is intended for the general purpose aerosol sampling, and a Nano-MOUDI (MSP Model 115) that is specifically designed for sampling aerosols in the size range down to 0.010 μm.
Welding particle metal content analysis
Mild and stainless steel welding particles were collected onto 5.0 mm polyvinyl chloride membrane filters in 37 mm cassettes during 30 min of welding. Metal analysis samples were collected separately from the samples used for free radical analysis. The particle samples were digested and the metals analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) by Clayton Group Services (A Bureau Vertis Company, Novi, MI) as coordinated with the Division of Applied Research and Technology (DART, Cincinnati, OH) according to NIOSH method 7300 modified for microwave digestion [50
]. Metal content of blank filters also was analyzed for control purposes.
Free radical measurements
ESR spin trapping was used to detect short-lived free radical intermediates. Hydroxyl radicals were measured using the addition-type reaction of a short-lived radical with a compound (spin trap) to form a relatively long-lived paramagnetic free radical product (spin adduct), which can then be studied using conventional ESR. For hydroxyl radical measurements on individual filter sizes, reactants were mixed in test tubes at a final volume of 1.0 ml of PBS in the presence of 1 mM H2
. The reaction mixture was then transferred to a quartz flat cell for ESR measurement. Experiments were performed at room temperature and under ambient air. The concentrations given in the figure legends are final concentrations. The intensity of the signal is used to measure the relative amount of short-lived radicals trapped, and the hyperfine couplings of the spin adduct are characteristic of the original trapped radicals. Spin trapping is the method of choice for detection and identification of free radical generation due to its specificity and sensitivity. All ESR measurements were conducted using a Bruker EMX spectrometer (Bruker Instruments Inc. Billerica, MA 01821, USA) and a flat cell assembly. Hyperfine couplings were measured (to 0.1 G) directly from magnetic field separation using potassium tetraperoxochromate (K3
) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) as reference standards [51
]. The relative radical concentration was estimated by multiplying half of the peak height by (ΔHpp
, where ΔHpp
represents peak-to peak width. The Acquisit program was used for data acquisitions and analyses (Bruker Instruments Inc. Billerica, MA 01821, USA).
Radicals trapped by bubbler
Freshly generated total welding particle reactivity was measured using a midget bubbler (Ace Glass, Vineland NJ) containing a reaction mixture made up of H2O2 (10 mM) DMPO (100 mM), and PBS to instantly trap generated radicals for ESR measurement. The midget bubbler was attached to a collection tube with a flow rate of 1 L/min positioned approximately 18 inches from the welding surface in order to directly collect the freshly generated fume. Fume was collected for 20 min. The reaction mixture was then immediately measured using ESR in order to determine .OH generation from freshly generated whole fume.
Grouped filter suspensions, were used for H2O2, lipid peroxidation, O2 consumption and DNA damage analysis. Suspensions were prepared by splitting the filters into three size groups. For this study the size groups were defined as ultrafine (0.01 - 0.056 μm), fine (0.10 - 1.0 μm) and coarse (1.8 - 18 μm) particles. The groups consisted of five filters each of which were placed in PBS and blended on ice into a fine slurry using a Tissue Tearor (Biospec Products Inc. Racine, WI). Pre- and post filter weights were used to prepare a final suspension of 1 mg/ml welding fume. The slurry was centrifuged, and the welding fume suspension was decanted from the filter pellet. A clean control suspension was also prepared at a ratio of 5 filters/1 ml PBS. Suspension not used immediately was aliquoted and frozen at -70°C.
Lipid peroxidation of RAW 264.7 mouse peritoneal monocytes was measured by using a colormetric assay for malondialdehyde (LPO-586 Oxis International Inc. Portland, OR, USA). A reaction mixture contained various size groups (refer to filter methods) of welding particle samples [250 μg/ml], H2
(1 mM) and 1 × 107
cells in a total volume of 1.0 ml PBS (pH 7.4). A Fenton reaction, FeSO4
(1 mM), H2
(1 mM) and 1 × 107
cells, was also carried out as a positive control. The mixtures were exposed for 1 h in a shaking water bath at 37°C. The measurement of lipid peroxidation is based on the reaction of a chromogenic reagent with malonaldehyde at 45°C [53
]. The absorbance of the supernate was measured at 586 nm.
H2O2 production was monitored using a Bioxytech quantitative hydrogen peroxide assay kit (H2O2-560, Oxis International Inc. Portland, OR, USA). Measurements were made on a system containing 5 × 106 RAW 264.7 mouse peritoneal monocytes/ml in pH 7.4 PBS and exposing them to the size groupings (refer to filter methods) of welding particle solution. Cells were exposed to welding particle solution [250 μg/ml], for 30 minutes in a 37°C incubator. Absorbance was monitored at a wavelength of 560 nm using a Spectra Max 250 multi-well plate reader (Molecular Devices, Sunnyvale, CA, USA).
Oxygen consumption measurements were carried out using a Gilson oxygraph (Gilson Medical Electronics, Middleton, WI). Measurements were made on a system containing 3 × 106 RAW 264.7 mouse peritoneal monocytes/mL and welding particle solution [500 μg/ml], in pH 7.4 phosphate buffer. The oxygraph was calibrated with media and equilibrated with known concentrations of oxygen.
DNA damage, Comet assay
The Comet assay was performed using methods described in the Trevigen (Gaithersburg, Md., U.S.A.) assay kit. A typical reaction mixture contained welding particle solution [250 μg/ml], H2
(1 mM), and 1 × 107
RAW 264.7 mouse peritoneal monocytes cells brought to a total volume of 1.0 mL in PBS (pH 7.4). Briefly, (all steps performed in the dark or low light conditions) RAW264.7 mouse cells were exposed to the various sized welding fume and incubated in media at 37 °C for 1 h. The cells were washed (2×) with PBS and combined with LMAgarose, then pipetted onto a Comet slide. The slide was placed in a refrigerator for 30 min, immersed in lysis solution, chilled for 60 min, and then immersed in alkaline solution for 55 min. Slides were placed in a horizontal electrophoresis chamber for 40 min at 300 mA. Slides were washed and SYBR green stain was added to each. Slides were visualized using fluorescence microscopy, with an image capturing system (Olympus AX70 and sample PCI, Compix, Cranberry Township, PA). A minimum of 50 cells were scored for each sample at 400× magnification. The distance between the edge of the head and the end of the tail was measured using an automated image analysis system (Optimas 6.51, Media Cybernetics Inc., Silver Spring, Md) [55
Data were expressed as mean ± standard error of the mean (SEM) (n = 4) for each group. One-way ANOVA test was performed using SigmaStat statistical software (Jandel Scientific, San Rafael, CA, USA) to compare the responses between treatments. Statistical significance was set at p < 0.05.