Toxicity of Diesel Engine Exhausts: Induction of a Pro-Inflammatory Response and Apoptosis in an In Vitro Model of Lung Slices in Biphasic Organotypic Culture

Precision-cut rat lung slices in organotypic culture, placed in a biphasic air/liquid system, are used for this study. This model allows to realize pathological and histochemical studies as well as cell and molecular biology investigations. Slices are exposed to a continuous flow of diluted diesel exhausts, with a pO₂ regulated at 20% in order to avoid hypoxia-induced effects. The exposure system allows to study concomitantly five exhaust concentrations coming from the same diesel engine, and also to evaluate the impact of particulate matter using a filter placed on the exposition vials. Precision lung slices are exposed for three or six hours to a whole or filtered diesel engine exhaust. Cytokine production (Tumor Necrosis Factor alpha, Interleukin 1 béta) in the culture medium is measured using E.L.I.S.A technique; DNA integrity is characterized by two distinct and complementary techniques: 1) nucleosome assay by an E.L.I.S.A method, 2) apoptosis phenomenon by a histochemical TUNEL method. After a three hour exposure, only TNF-α is detected and increased in culture medium of lung slices exposed to a whole diesel exhaust. In the same conditions, slice nucleosome level increases in a dose-dependant way. For TUNEL method, apoptotic cells are detected after a six hour exposure followed by an incubation period of 18 hours in controlled atmosphere (5% CO₂/ 95% O₂). Under these conditions, apoptotic nuclei are more frequent after diesel exhaust exposure than in control slices.

In conclusion, exposure to whole diesel exhaust induces an inflammatory response and DNA alterations in lung tissue. These alterations are reduced by filtration thus pointing out the important role of the diesel exhaust particulate matter.