Synergistic effect of co-exposure to carbon black and Fe(2)O(3 )nanoparticles on oxidative stress in cultured lung epithelial cells

BACKGROUND: There is a need to better understand synergism in the biological effects of particles composed of multiple substances. The objective of this study was to determine if the oxidative stress in cultured cells caused by co-exposure to carbon black and Fe(2)O(3 )nanoparticles was significantly greater than the additive effects of exposure to either type of particles alone; and to determine a possible cause for such synergistic effect if one was found. Cultured A549 human lung epithelial cells were exposed to (1) carbon black nanoparticles alone, (2) Fe(2)O(3 )nanoparticles alone, and (3) both types of particles simultaneously. Protein oxidation, lipid peroxidation, and cellular uptake of Fe in these cells were measured after 25 hours of exposure. The reduction of solubilized Fe(3+ )by the carbon black nanoparticles was measured separately in a cell-free assay, by incubating the carbon black and the Fe(2)O(3 )nanoparticles in 0.75 M sulfuric acid at 40°C and measuring the amount of reduced Fe(3+ )at different time points up to 24 hours. RESULTS: Cells exposed to carbon black particles alone did not show protein oxidation, nor did the cells exposed to Fe(2)O(3 )particles alone, relative to the control. However, cells co-exposed to both carbon black and Fe(2)O(3 )particles showed up to a two-fold increase in protein oxidation relative to the control. In addition, co-exposure induced significant lipid peroxidation, although exposure to either particle type alone did not. No significant difference in cellular iron uptake was found between single exposure and co-exposure, when the Fe(2)O(3 )dosing concentration was the same in each case. In the cell-free assay, significant reduction of Fe(3+ )ions by carbon black nanoparticle was found within 2 hour, and it progressed up to 24 hours. At 24 hours, the carbon black nanoparticles showed a reductive capacity of 0.009 g/g, defined as the mass ratio of reduced Fe(3+ )to carbon black. CONCLUSION: Co-exposure to carbon black and Fe(2)O(3 )particles causes a synergistic oxidative effect that is significantly greater than the additive effects of exposures to either particle type alone. The intracellular redox reaction between carbon black and Fe(3+ )is likely responsible for the synergistic oxidative effect. Therefore elemental carbon particles and fibres should be considered as potential reducing agents rather than inert materials in toxicology studies. Acidified cell organelles such as the lysosomes probably play a critical role in the solubilization of Fe(2)O(3). Further research is necessary to better understand the mechanisms.