In vitro and in vivo assessment of pulmonary risk associated with exposure to combustion generated fine particles

Abstract

Strong correlations exist between exposure to PM_2.5 and adverse pulmonary effects. PM_2.5 consists of fine (≤2.5 μm) and ultrafine (≤0.1 μm) particles with ultrafine particles accounting for >70% of the total particles. Environmentally persistent free radicals (EPFRs) have recently been identified in airborne PM_2.5. To determine the adverse pulmonary effects of EPFRs associated with exposure to elevated levels of PM_2.5, we engineered 2.5 μm surrogate EPFR-particle systems. We demonstrated that EPFRs generated greater oxidative stress in vitro, which was partly responsible for the enhanced cytotoxicity following exposure. In vivo studies using rats exposed to EPFRs containing particles demonstrated minimal adverse pulmonary effects. Additional studies revealed that fine particles failed to reach the alveolar region. Overall, our study implies qualitative differences between the health effects of PM size fractions.

Introduction

Epidemiological studies demonstrate a clear correlation between exposure to particulate matter (PM) and adverse pulmonary effects such as asthma exacerbation (Delfino et al., 2004). Airborne PM is generated by a variety of sources including industrial processes and combustion of biomass and fossil fuels (Zheng et al., 2002). Recently, we reported the presence of environmentally persistent free radicals (EPFRs) associated with ambient PM_2.5 samples collected from different locations across the United States (Dellinger et al., 2001). In combustion processes, high-temperatures initiate a cascade of chemical reactions which form phenoxyl- and semiquinone-type radicals depending on the precursors present (Lomnicki et al., 2008a) that are stabilized and resistant to oxidation when associated with metal oxide containing particles and thus persist in the environment (i.e. EPFR). EPFRs are of significant interest due to their redox potential and ability to produce reactive oxygen species (ROS) in biological systems (Lomnicki et al., 2008b).

Although the adverse pulmonary effects of ambient PM have been extensively studied by epidemiologists, many of these studies were performed with a mixture of particles, collected from ambient air and were of various sizes and chemical compositions. Thus, the biological impact related to a specific chemical or particle size has remained an enigma. We have developed a surrogate model for EPFRs using 2-monochlorophenol (MCP) and particles having a median diameter of 2.4 μm. The process of EPFR synthesis is initiated by simple physisorption of the molecular precursors on the surface, followed by chemisorption at a metal oxide (e.g. Fe₂O₃ or CuO) surface site at high-temperatures (230 °C) like those encountered in the post-flame cool-zone of combustors where pollutants like dioxins are known to form. The adsorbate then reduces the metal oxide to form a EPFR (Lomnicki et al., 2008b).

We chose CuO as the metal oxide in our model system because it is present in relatively high concentrations in biomass such as woody wastes and debris as well as cigarette smoke (Wasson et al., 2005, Neuberger et al., 2009). 2-MCP was selected because it is typically produced from thermal treatment of wastes and is able to chemically react with CuO under combustion-like conditions. A sample of CuO/silica was exposed to 2-MCP at 230 °C which resulted in the formation of the persistent 2-MCP radical on the surface of the CuO/silica. CuO/silica and silica/MCP serve as control particles to understand the roles of CuO and MCP, in the biological responses following exposure. Our hypothesis was that an EPFR (e.g. silica/CuO/MCP), which undergoes redox cycling and produces ROS, is able to generate more oxidative stress and induce greater damage to human epithelial cells than the silica particles containing CuO or 2-MCP alone.

Excluding particle dose, time points, and end points for assessing toxicity; there are many other inherent limitations to in vitro systems. First, epithelial cells represent only a fraction of all of the cells present in the respiratory system. Second, epithelial cells lack many of the defensive capabilities of a lung in vivo including a blood supply, immune system, endogenous buffering system, and other biologically relevant factors. Third, the ability of the surrogate pollutant/particle systems to reach the alveolar region of the lungs remains controversial (Ferin et al., 1992, Kreyling et al., 2006). Thus, we further hypothesized that fine combustion generated particle surrogates (≤2.5 μm in spherical diameter) would produce minimal adverse effects on pulmonary function.

To investigate the intrinsic ability of fine particles containing EPFR to decrease cell viability and generate oxidative stress in respiratory epithelial cells, an in vitro model was employed. Human laryngeal epithelial cells (HEp-2) were chosen, since they are used in many pulmonary toxicological assays (Rudolf et al., 2001, Kvolik et al., 2005) and represent target cells which are usually subjected to significant amounts of airborne particles. For the evaluation of the in vivo effects of an EPFR, we employed neonatal rats, which unlike adults, have immature lungs characterized by saccular structure, thick alveolar walls, inadequate pulmonary capillary network and limited gas-exchange capabilities and are very sensitive to air pollutants (Hsia et al., 2004). The development of airway inflammation and oxidative stress in neonatal pulmonary tissue as result of exposure to air pollutants such as PM_2.5 can lead to the induction of airway remodeling and alter pulmonary function (Jeffery, 2001, Henson et al., 2006). Accordingly, we have assessed the impact of EPFRs in both an in vitro culture system and an in vivo system following inhalation exposure in neonatal rats.