Oxidative-stress potency of organic extracts of diesel exhaust and urban fine particles in rat heart microvessel endothelial cells

Abstract

Exposure to fine particulate materials is associated with an increase in mortality rate of cardiovascular diseases. Particles deposited in the lung may affect the vascular system both directly (leaching of soluble components from particles) and indirectly (via cytokines and mediators). The present study addressed cytotoxicity and oxidative stress potency of organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) in rat heart microvessel endothelial (RHMVE) cells. The LC₅₀ values of OE-DEP and OE-UFP were calculated to be 17 and 34 μg/ml, respectively, suggesting that OE-DEP was more cytotoxic than OE-UFP. The viability of OE-DEP- and OE-UFP-exposed cells was ameliorated by N-acetyl-l-cysteine (NAC). The cell monolayer was exposed to 0 (control), 1, 3, and 10 μg/ml OE-DEP for 6 h and mRNA levels of antioxidant enzymes such as heme oxygenase-1 (HO-1), thioredoxin peroxidase 2 (TRPO), glutathione S-transferase P subunit (GST-P), and NADPH dehydrogenase (NADPHD) were quantitated by northern analysis. All those mRNA levels increased dose-dependently with OE-DEP and HO-1 mRNA showed the most marked response to OE-DEP. mRNA levels of those antioxidant enzymes and heat shock protein 72 (HSP72) in OE-DEP-exposed cells were higher than those of OE-UFP-exposed cells as compared at the same concentration. The transcription levels of HO-1 and HSP72 in OE-DEP- and OE-UFP-exposed cells were also reduced by NAC. Those results suggest that the organic fraction of particulate materials in the urban air has a potency to cause oxidative stress to endothelial cells and may be implicated in cardiovascular diseases through functional changes of endothelial cells.

Introduction

Several epidemiological studies have shown that particulate air pollution is associated with cardiopulmonary and lung cancer motility (Dockery et al., 1993, Pope et al., 1995). Ambient particulate materials contain sulfate, heavy metals, and organic compounds and those harmful substances can cause cardiopulmonary diseases and increase airway sensitivity (Amdur et al., 1978, Busch et al., 1984, Frampton et al., 1999, Gordon et al., 1998, Kennedy et al., 1998, Kodavanti et al., 2000a, Kodavanti et al., 2000b). It has also been shown that urban particulate materials induced proinflammatory cytokines in human bronchial epithelial cells (Fujii et al., 2001, Reibman et al., 2002), lung epithelial cells (Jimenez et al., 2002), and macrophages (Becker et al., 1996, van Eeden et al., 2001). The interaction of macrophages with epithelial cells amplifies the cytokine production in those cells (Tao and Kobzik 2002).

Diesel exhaust particles (DEP) form a large constituent of ambient urban fine particles (UFP) and comprise of carbonaceous cores, aliphatic and aromatic hydrocarbons, quinones, aldehydes, and heterocyclic organic compounds (Higgins et al., 1979, Reynolds and Richards, 2001, Schuetzle, 1983). Inhalation and intratracheal instillation of DEP and organic extracts of DEP (OE-DEP) have been shown to cause lung inflammation, aggravation of asthmatic symptom, lung cancer, and electrocardiographic alterations (Minami et al., 1999, Miyabara et al., 1998, Takano et al., 1998, Takano et al., 1997, Whitekus et al., 1999). OE-DEP induces heat shock proteins and apoptosis in macrophages (Hiura et al., 2000, Li et al., 2000), cytokine expression in bronchial epithelial cells (Kawasaki et al., 2001, Takizawa et al., 1999), nitric oxide production in endothelial cells (Bai et al., 2001, Muto et al., 1996). It has been shown that DEP produced superoxide and hydroxyl radicals (Sagai et al., 1993) and were implicated in 8-hydroxy-deoxyguanosine and tumor formation in the mouse lung (Ichinose et al., 1997). It is plausible that UFP causes oxidative stress to the lung as well as DEP.

Fine particulate materials penetrate deep in the lung and deposit in the alveolar space. Thus, pulmonary epithelial cells and alveolar macrophages are primary targets of inhaled fine particulate materials. However, it is not clear why pulmonary deposition of particulate materials leads to an increase in mortality of cardiovascular diseases as reported in epidemiological studies. There are several mechanisms to explain the particulate material-induced cardiovascular diseases; (1) particulate materials deposited in the lung exacerbate pulmonary lesions and increase blood pressure of pulmonary circulation, (2) cytokines or mediators released from particle-loaded lung tissue affect cardiopulmonary function, (3) toxic organic compounds or heavy metals leach from the particles, reach to the endothelium, and damage the vascular systems.

Recently we reported that mRNA levels of antioxidant enzymes selectively increased in OE-DEP-exposed rat alveolar macrophages using cDNA microarray and northern analysis (Koike et al., 2002). Short-term inhalation exposure to concentrated ambient particles (CAPS) has been shown to cause oxidative stress and increase antioxidant enzymes such as superoxide dismutase and catalase in the rat lung (Gurgueira et al., 2002). We postulated that organic materials leach from DEP and UFP in the lung and may directly affect vascular system. In the present study we report cytotoxicity and oxidative stress of OE-DEP and DE-UFP on endothelial cells.