Diesel and biodiesel exhaust particle effects on rat alveolar macrophages with in vitro exposure

Abstract

Combustion emissions from diesel engines emit particulate matter which deposits within the lungs. Alveolar macrophages (AMs) encounter the particles and attempt to engulf the particles. Emissions particles from diesel combustion engines have been found to contain diverse biologically active components including metals and polyaromatic hydrocarbons which cause adverse health effects. However little is known about AM response to particles from the incorporation of biodiesel. The objective of this study was to examine the toxicity in Wistar Kyoto rat AM of biodiesel blend (B20) and low sulfur petroleum diesel (PDEP) exhaust particles. Particles were independently suspended in media at a range of 1-500μgmL(-1). Results indicated B20 and PDEP initiated a dose dependent increase of inflammatory signals from AM after exposure. After 24h exposure to B20 and PDEP gene expression of cyclooxygenase-2 (COX-2) and macrophage inflammatory protein 2 (MIP-2) increased. B20 exposure resulted in elevated prostaglandin E2 (PGE2) release at lower particle concentrations compared to PDEP. B20 and PDEP demonstrated similar affinity for sequestration of PGE2 at high concentrations, suggesting detection is not impaired. Our data suggests PGE2 release from AM is dependent on the chemical composition of the particles. Particle analysis including measurements of metals and ions indicate B20 contains more of select metals than PDEP. Other particle components generally reduced by 20% with 20% incorporation of biodiesel into original diesel. This study shows AM exposure to B20 results in increased production of PGE2in vitro relative to diesel.

Keywords: Alveolar macrophages; Biodiesel exhaust; Diesel exhaust; Prostaglandin E(2).

Figure 1

Density of 2×10⁴ WKY AM incubated 24h with (A) vehicle, (B) B20 at 10µg/mL and (C) B20 at 1µg/mL. All images are of fixed and H&E stained cells. Images taken with Nikon eclipse E600 at 20× magnification. Solid arrow (←) points to unbound B20 (◂----) indicates B20 within or attached to the surface of the AM.

Figure 2

Cell cytotoxicity of AM after 24h exposure to 1–500µg/mL of B20 or PDEP measured by LDH release. Each group represents mean ± SE (n=6). Exposure to B20 or PDEP shows no significant cell toxicity as fold change relative to media control.

Figure 3

Gene expression of COX-2 and MIP-2 in AM exposed for 24h to B20 and PDEP. COX-2 expression is significantly increased in both B20 and PDEP at 100µg/mL; B20 *P< 0.05 and PDEP ^#P<0.001. MIP-2 expression with B20 increases with dose but not significant and PDEP at 100µg/mL is statistically significant *P<0.05. Each group represents mean ± SE (n=10).

Figure 4

AM PGE₂ release following 24h exposure to PDEP and B20 was quantified by ELISA. Each group represents mean ± SE of 3–12 animals. B20 concentration of 10µg/mL is statistically significant with reference to media control *P<0.05.

Figure 5

B20 and PDEP binding of hot (³H-PGE₂) and cold (PGE₂) in a cell free system. Particles were suspended in RPMI media with 2.5% FBS for 24h, particles were centrifuged and supernatant was collected for standard scintillation counting. B20 at both 100µg/mL and 500µg/mL and PDEP at 500µg/mL are statistically significant *P < .001 from media control.