Development of a new in vitro system for continuous in vitro exposure of lung tissue to complex atmospheres: Application to diesel exhaust toxicology J.-P. Morin 1 , F. Fouquet 1 , C. Monteil 1 , E. Le Prieur 1 , E. Vaz 2 and F. Dionnet 3 1 VACOMED- INSERM E9920, UFR Me¨ decine Pharmacie, Universite¨ de Rouen; 2 Service de Me¨ decine Le¨ gale CHU Rouen; 3 CERTAM/CORIA ^ CNRS UMR 6614, Universite¨ de Rouen, France Received 28 January 1999; accepted 23 March 1999 Keywords: diesel exhaust, in vitro, lung, organotypic culture, slices, toxicology Abstract The purpose of this study was the development of a new incubation system that can allow continuous exposure of lung tissue to complex atmospheres as a tool for the assessment of aerial environmental lung toxicology. To assess the pertinence of this new exposure system, we studied the impact of diesel engine exhausts as a complex atmosphere containing both gaseous and particulate fractions and have been able to discriminate between the toxicological impacts of the gaseous phase and particulate matter from diesel exhausts. Continuous £ow-through rotating chambers with controlled pO 2 , pCO 2 , and hygrometry have been designed in which lung slices are positioned in rolling inserts that allow free access of atmosphere to the exposed lung tissue. Under control conditions, cell viability was preserved for at least 48 h as assessed by intracellular ATP, GSH, and K + levels and slice O 2 consumption levels. Short-term exposure (1 h) to diesel whole exhausts did not a¡ect intracellular potassium or slice O 2 consumption, while intracellular ATP and GSH levels were markedly decreased. Exposure to ¢ltered exhausts showed less marked e¡ects on both ATP and GSH levels. Superoxide dismutase activity was decreased in a similar way by both total and ¢ltered exhausts while Se + -dependent glutathione peroxidase activity was induced by ¢ltered exhausts to a larger extent than after total exhaust exposure, showing di¡erent response patterns of lung tissue after exposure to whole or ¢ltered exhausts. In conclusion, this newly designed model opens a promising area in in vitro environmental lung toxicology testing. Abbreviations: GSH, glutathione; GPX, glutathione peroxidase; QO 2 , oxygen consumption Introduction Respiratory toxicity is a major health concern. The total area of pulmonary epithelium that is exposed to air is estimated at *80^100 m 2 in human beings and *8m 2 in the rat (Swemer, 1970). The contribution of nonalveolar surface area to these values appears to be of very minimal importance, especially in the rat, in which the bronchiolar tree is underdeveloped compared to humans. This epithelium is the site of numerous physiological functions but also represents, owing to its huge surface area, a potential route for environmentally hazar- dous chemical e¡ects. Inhalation toxicology in vitro is a very complex subject that has most Cell Biology and Toxicology . 1999; 15: 143^152. # 1999 Kluwer Academic Publishers. Printed in the Netherlands