Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine particles

Abstract

In ambient aerosols, ultrafine particles (UFP) and their agglomerates are considered to be major factors contributing to adverse health effects. Reactivity of agglomerated UFP of elemental carbon (EC), Printex 90, Printex G, and diesel exhaust particles (DEP) was evaluated by the capacity of particles to oxidize methionine in a cell-free in vitro system for determination of their innate oxidative potential and by alveolar macrophages (AMs) to determine production of arachidonic acid (AA), including formation of prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄), reactive oxygen species (ROS), and oxidative stress marker 8-isoprostane. EC exhibiting high oxidative potential induced generation of AA, PGE₂, LTB₄, and 8-isoprostane in canine and human AMs. Printex 90, Printex G, and DEP, showing low oxidative capacity, still induced formation of AA and PGE₂, but not that of LTB₄ or 8-isoprostane. Aging of EC lowered oxidative potential while still inducing production of AA and PGE₂ but not that of LTB₄ and 8-isoprostane. Cellular ROS production was stimulated by all particles independent of oxidative potential. Particle-induced formation of AA metabolites and ROS was dependent on mitogen-activated protein kinase kinase 1 activation of cytosolic phospholipase A₂ (cPLA₂) as shown by inhibitor studies. In conclusion, cPLA₂, PGE₂, and ROS formation was activated by all particle types, whereas LTB₄ production and 8-isoprostane were strongly dependent on particles' oxidative potential. Physical and chemical parameters of particle surface correlated with oxidative potential and stimulation of AM PGE₂ and 8-isoprostane production.

Introduction

Worldwide, acute exposure to inhaled ambient particles has been found to be associated with adverse health effects. The fraction of ultrafine particles in the ambient aerosol is considered a major factor contributing to adverse health effects, including pulmonary and cardiovascular diseases [1], [2], [3], [4]. The size distribution of particles at urban sites indicates that the ultrafine fraction (<0.1 μm) represents about 72% of the number concentration of particles in the ambient aerosol, whereas their mass concentration is negligible (1.1%) compared to the fine particle fraction (<2.5 μm) [5]. Due to their very small diameters (<0.1 μm), the ultrafine particles are predominantly deposited in the periphery of the lungs and interact with cells of the alveolar region such as alveolar macrophages (AMs) and epithelial type I and II cells. The biologic reactivity of ultrafine particles is supposed to be determined by their large specific surface area. Our previous study with AMs indicated that the effect of ultrafine particles of elemental carbon (EC) and of titanium dioxide (TiO₂) on lipid mediator generation is determined by the specific surface area rather than the mass concentration of the particles [6]. Oberdörster et al. [7] have shown that instillation of ultrafine particles of TiO₂ (diameter 21 nm, surface area 50 m²/g) into the lungs of rats elicited a stronger inflammatory response than that of fine TiO₂ particles (diameter 250 nm, surface area 6.5 m²/g). These studies reveal that the toxicity of the particles is related to their surface area. Findings of Brown et al. [8] confirmed that increased numbers of neutrophils in the bronchoalveolar lavage fluid induced by instilled ultrafine and fine polystyrene particles in rat lungs correlated with the surface area of these particles. Furthermore, there is evidence that ultrafine particles elicit oxidative stress [8], [9]. However, the molecular mechanisms underlying these particle-induced effects are still not well known.

A recent study has shown that AMs obtained from children contained ultrafine carbonaceous particles singly but also as aggregates. The percentage of particle-containing AMs was higher in children whose parents lived on a main road compared to those living in a quiet residential area [10]. Furthermore, ultrafine particles have also been found in AMs from healthy nonsmoking adults, suggesting an environmental exposure to ultrafine particles [11]. These observations indicate that AMs are relevant target cells for ultrafine particles.

For the present in vitro study we hypothesized that the oxidative potential of ultrafine particles reflects their surface reactivity, which is decisive for their biologic impact. We therefore analyzed the oxidative potential of various ultrafine carbon particles in a cell-free in vitro system as a measure for their surface reactivity. In addition, we evaluated in primary canine or human AMs their biologic response to ultrafine particles especially focusing on lipid mediator synthesis and lipid peroxidation indicating oxidative stress. AMs as part of the primary pulmonary defense system produce pro- and anti-inflammatory mediators, including metabolites of arachidonic acid such as leukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂), after interference with particulate matter [6]. As models for ultrafine particles we used agglomerates of ultrafine particles of EC, Printex 90, Printex G, and diesel exhaust particles (DEP). EC consists of spherical primary particles with a uniform size of a few nanometers (5–10 nm), which combine to randomly form agglomerates and simulate the carbonaceous matrix of diesel exhaust particles [12], [13]. Printex 90 and Printex G are both ultrafine carbonaceous particles with different specific surface areas. They are commercially available and used as carbon black particles for various technical applications. DEP (Standard Reference Material 1650a) represents ultrafine environmental particles [12].