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

Abstract

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 260 nm), and quartz (up to 0.78 μg/mm²) 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 (2 mm). 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.

Introduction

Recently, there has been considerable attention paid to the potential pathogenic effects of ultrafine and/or nanometer (<100 nm diameter) particles which are readily inhaled and deposited throughout the lung. Ultrafine particles are increasingly synthesized and utilized industrially, for ‘high-tech’ applications which require properties such as improved hardness and reduced internal friction (Pui and Chen, 1997). Interest in the potential adverse health effects of ultrafine particles was fuelled by observations that ultrafine TiO₂ (Ferin et al., 1992) and ultrafine carbon black (Li et al., 1996) are more inflammogenic in rat lungs than fine particles of the same material. In addition, much attention has focused on the pathogenic effects of the ultrafine component of environmental particles, PM₁₀ (particulate matter<10 μm diameter) since it has been hypothesized that the ultrafine component mediates the harmful effects of PM₁₀ in susceptible individuals (Oberdorster, 1995; Seaton et al., 1995).

Many respirable particles have been shown to induce oxidative damage (Howard et al., 1996; Lenz et al., 1992; Li et al., 1996) and/or to demonstrate free radical activity in cell free systems (Gilmour et al., 1995, Gilmour et al., 1997; Howden and Faux, 1996). It is suggested that liberation of free radicals at the surface of the particles, either spontaneously or during phagocytosis by macrophages, leads to an increased oxidative stress culminating in an increase in the inflammatory response and injury to the surrounding cells, particularly the epithelium (Li et al., 1996). The exact mechanism by which ultrafine particles induce a greater inflammatory response than fine particles of an identical chemical composition remains uncertain. However, ultrafine particles possess both a greater particle number and total surface area per unit mass of fine particles. Hence, we hypothesize that the harmful effects of ultrafine particles may be due to the comparatively high number of particles exposed to the cells and/or due to their greater surface area available for the production of free radicals (Gilmour et al., 1997; Oberdorster, 1996).

The present in vitro study has compared the potential differences in the ability of fine carbon black (CB) and ultrafine carbon black (uf CB) (Plate I) to produce oxidative stress on exposure to the human alveolar type II cell line A549. In this paper, the term ‘fine’ is used to describe respirable particles above the ultrafine size range (i.e. >100 nm). We have compared the effects of these particles with quartz (Sikron F600) which is also thought to have oxidant properties (Castranova, 1994). Damage to the lung parenchyma by quartz may occur through its oxidant properties or though direct toxicity (Lapp and Castranova, 1993). In this study, the relative toxicities of the three particles have been compared. In addition, we have assessed the effect of low relatively non-toxic doses (0.39 and 0.78 μg/mm²) of all three particles on the reduction of MTT in A549 cells as a measure of the metabolic competence. We have investigated the role played by the hydroxyl radical using a specific hydroxyl radical scavenger, mannitol, as well as by assessing the effects on intracellular levels of the important intracellular antioxidant reduced gluthathione (GSH) and its oxidized form glutathione disulfide (GSSG).