Lung Toxicity Profile of Inhaled Copper-Nickel Welding Fume in A/J Mice

Data Collection Methods

Animals

• Male A/J mice aged 4-6 weeks were house in a specific pathogen-free, environmentally controlled facility.

Welding Fume Inhalation Exposure System

• The automated robotic welder continuously generated welding fumes by welding beads onto ¼ inch thick plates of mild steel.
• The resulting fume was carried into a whole-body exposure chamber through a flexible tube by maintaining the chamber at a negative pressure. Particle concentrations and gas generation within the exposure chamber were continuously monitored.
• Welding fume characterization was conducted on filters present in the exposure chamber that collected particles that were generated.

Whole Lung Metal Analysis

• Weight-matched A/J mice were exposed by whole-body inhalation in individual cages to Cu-Ni welding aerosols for 4 hours or filtered air.
• Immediately following exposure, mice were euthanized. Whole lungs were excised, trimmed, and lyophilized.
• The freeze-dried tissue was weighed then acid digested.
• ICP-AES was used to determine the amount of Al, Ba, Ca, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Ni, P, Pb, Sr, Ti, V, Zn and Zr present in the lung.

Assessment of Lung Toxicity

• Mice were exposed by whole-body inhalation to Cu-Ni welding fume aerosols or filtered air for 2 [low deposition (LD)] or 4 [high deposition (HD)] hours/day for 10 days.
• Before the start of the exposure mice were weight-matched then weighed biweekly and again at the terminal sacrifice of 1,7,28, and 84 days after the 10 day exposure.
• Mice were euthanized and bronchioalveolar lavage (BAL) fluid was collected through a cannula placed in the trachea. The thorax was massaged as phosphate buffered saline (PBS) was instilled into the lungs. After 10 seconds, the BAL fluid was withdrawn and collected.
• This process was then repeated 3 times and this second fraction was collected separately.
• The first fraction of BAL fluid was used to measure LDH activity
• Cells of both fractions were combined for cell counts and differential staining. Total cell numbers were determined using a hemocytometer.
• For cell differentials, cells were plated onto glass slides, stained, and coverslipped. A minimum of 300 cells/slide, consisting of macrophages, lymphocytes, and polymorphonuclear leukocytes were identified.

Macrophage Functional Assay

• Lung macrophages harvested by BAL at 1, 7, and 28 days post-exposure were challenged with Escherichia coli (E. coli) GFP for 2 hours. The uptake of E.coli by macrophages was quantified by flow cytometry.