Development of novel diesel exhaust particle aerosolization and deposition methods for in vitro toxicology studies.

摘要

The influence of diesel exhaust particles (DEP) on the lungs and heart is currently a topic of great interest in inhalation toxicology. Epidemiologic data and animal studies have implicated airborne particulate matter and DEP in increased morbidity and mortality due to a number of cardiopulmonary diseases including asthma, chronic obstructive pulmonary disorder, and lung cancer. The pathogeneses of these diseases are being studied using cell culture techniques. The current prevalent methodology for evaluating the effects of DEP on cells in culture (exposure to particles suspended in culture media) does not reflect lung delivery of these materials to human lungs. Consequently, the effects of DEP exposure may be difficult to elucidate. A method of in vitro DEP deposition that more closely mimics the in vivo dosing and deposition would be a desirable and useful alternative. It was proposed that DEP delivered as aerosols to cells in culture is physiologically relevant and would result in measurable inflammatory responses of toxicological significance. The specific aims of this dissertation were addressed by: (i) generating DEP aerosols in a size range relevant to lung deposition and characterizing those aerosols; (ii) formulating methods for deposition of those aerosols onto cells in culture; (iii) studying the effects of DEP exposure following this type of deposition on reactive oxygen species formation, the production of pro-inflammatory mediators, and viability. Several commercial particle sizing instruments and a novel device were used to deposit DEP aerosols onto the apical surface of Calu-3, A549, and/or 16HBE14o- cells by inertial impaction. Deposition of DEP aerosols onto A549 cells in the novel device elicited a cellular response that precludes their use in studies of toxicity. Deposition by inertial impaction did not appear to affect Calu-3 cells, and the response of these cells to exposure to DEP aerosols was shown to be different from the response to exposure to DEP as a media suspension. Methods of re-dispersing aerosols of DEP, sampling onto cell surfaces, and studying their effect were established and demonstrate the utility and relevance of this approach.