The role of oxidative stress in the prolonged inhibitory effect of ultrafine carbon black on epithelial cell function.

Respired ultrafine particles induce a greater inflammation in rat lungs than fine particles; we have hypothesized that this is due to their comparatively huge number and surface area for the production of free radicals. We tested this hypothesis by studying the effects of fine and ultrafine (uf) carbon black (CB) particles in comparison with quartz on A549 human type II alveolar epithelial cells, particularly with respect to the oxidative properties of these particles. Treatment with fine CB (diameter 260nm), and quartz (up to 0.78mug/mm(2)) for 24 hours significantly (P<0.05) decreased the A549 cells metabolic competence, as measured by the ability to reduce MTT to a formazan product. The inhibitory effects of uf CB only became significantly different (P<0.05) relative to the control at 48 hours, by which time the effects of fine CB and quartz were no longer significant. The inhibition of MTT reduction by uf CB was prevented by the hydroxyl radical scavenger mannitol (2mm). In addition, measurement of reactive oxygen species production using supercoiled plasmid DNA showed that uf CB exhibited significantly more free radical activity than fine CB (P<0.05). In the absence of serum, uf CB depleted reduced glutathione at 6 hours (P<0.008). In contrast, CB did not significantly alter reduced or oxidized glutathione. Hence, compared with fine CB, uf CB exhibited greater free radical activity, greater inhibition of the reduction of MTT at 48 hours (prevented by mannitol) and a depletion of reduced glutathione. These results suggest that uf CB induces a greater oxidative stress than fine CB, and that this may play a role in the toxicological effects of this ultrafine particle.