Toxicity of diesel engine exhausts in an in vitro model of lung slices in biphasic organotypic culture: induction of a proinflammatory and apoptotic response.

摘要

Precision-cut rat lung slices in organotypic culture placed in a biphasic air/liquid system were used for this study. This model allowed pathological as well as cellular and molecular biology investigations to be carried out. Slices were exposed to a continuous flow of diluted diesel exhaust, with a pO2 adjusted to 20% to avoid hypoxia-induced effects. The exposure system allowed five exhaust concentrations from the same diesel engine to be studied concomitantly, and also allowed the impact of removing the particulate matter using a filter cap on the exposure vials to be evaluated. Lung slices were exposed for 3 or 6 h to whole or filtered diesel exhaust. DNA integrity was characterized by two different techniques: (1) an ELISA for the determination of nucleosomes, and (2) the histochemical TUNEL method. By the TUNEL method, apoptotic cells were detected after a 6-h exposure followed by an incubation period of 18 h in a controlled atmosphere comprising 5% CO2/95% O2. Under these conditions, apoptotic nuclei were more frequent in slices exposed to diesel exhaust than in control slices. Cytokine production (tumor necrosis factor alpha, interleukin-1beta) in the culture medium was measured using an ELISA technique. After a 3-h exposure only TNF-alpha was detected and increased in the culture medium of lung slices exposed to diesel exhaust. Under the same conditions, nucleosome levels in the slices increases in a dose-dependent way. In conclusion, whole diesel exhaust induced an inflammatory response and DNA alterations which were reduced by filtration, thus indicating the important role of the particulate matter in diesel exhaust.