Environmental Fine Particulate Matter (PM 2.5) Activates the RAW 264.7 Macrophage Cell Line Even at Very Low Concentrations as Revealed by 1 H NMR Maria Teresa Santini,* ,†,‡ Gabriella Rainaldi, †,‡ Antonella Ferrante, † Rocco Romano, ‡,§ Stefania Clemente, § Andrea Motta, | Barbara De Berardis, † Maria Balduzzi, ⊥ Luigi Paoletti, † and Pietro Luigi Indovina ‡,§ Laboratorio di Ultrastrutture, Istituto Superiore di Sanita ` , Viale Regina Elena 299, 00161 Rome, Italy, Istituto Nazionale per la Fisica della Materia, Unita ` di Napoli and Dipartimento di Scienze Fisiche, Universita ` di Napoli “Federico II”, Complesso Universitario Monte S. Angelo, Via Cinthia, 80126 Naples, Italy, Istituto di Chimica Biomolecolare del Consiglio Nazionale delle Ricerche, Comprensorio Olivetti, Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy, and UTS Biotecnologie, Protezione dell’Ambiente e degli Ecosistemi, ENEA, Via Anguillarese 301, 00060 Rome, Italy Received June 11, 2003 Because of the association between inhalation of airborne particulate matter (PM) and human respiratory and cardiovascular disease, it is necessary to understand the tissue damage induced by these particles. One of the cell types principally involved in the body’s reaction to PM are macrophages, which remove particles in the airway passages and the lungs through phago- cytosis. In fact, when macrophages are exposed to a toxic agent such as PM, they undergo a series of changes (including variations in morphology, an increase in glycolysis, and consequent lactate production and the release of cytokines such as interleukin-6 and tumor necrosis factor- R) necessary to transform them from “resting” to “activated” macrophages. Because 1 H NMR is extremely useful in monitoring, noninvasively, macrophage metabolism and because this technique has never been utilized to examine macrophage activation after exposure to PM, it was the purpose of the present study to investigate the effects of PM exposure on the RAW 264.7 stabilized macrophage cell line using 1 H NMR spectroscopy. PM with a diameter <2.5 µm (PM 2.5) was utilized because a closer association to mortality and adverse respiratory health effects has been found with this fraction than with particles of a larger size. Measurements were conducted on whole cells at both 500 and 700 MHz as well as on perchloric acid extracts at 700 MHz. Significant variations in numerous metabolites were seen at very low concentrations of PM 2.5. Many of these changes point to activation of RAW 264.7 macrophages even at doses of PM 2.5 much lower than those commonly employed in cell studies. These results are particularly significant since the same concentrations of PM did not induce changes in morphology and release of cytokines in these cells. Therefore, 1 H NMR spectroscopy is an extremely sensitive probe in observing subtle variations in macrophages after exposure to PM 2.5. Introduction Airborne particulate matter (PM) is composed of complex aggregates of inorganic materials, salts, and organic compounds (1, 2). A direct association has been made between inhalation of these particles and human respiratory and cardiovascular disease (3-6). Because of the gravity of the diseases involved and because human exposure to PM is increasing worldwide, it is necessary to better comprehend the complex toxicological mecha- nisms at the basis of PM-induced tissue damage and improve current knowledge of the response of target cells to airborne particles. One of the cell types principally involved in the body’s reaction to PM are macrophages. In fact, during respira- tion, PM enters the upper airway passages and the lungs. Because PM is potentially dangerous to the organism, macrophages must remove it through phagocytosis in order to protect the organism from contamination. In fact, these cells are responsible for phagocytizing foreign materials that enter the lungs and for keeping this organ clean (7). In addition, macrophages also play a pivotal role in both acute and chronic pulmonary and bronchial inflammation since they are recruited at sites of inflam- mation by the release of chemicals (e.g., cytokines). It is this continuous production of cytokines that leads to chronic inflammation and tissue damage (8). When macrophages are exposed to a toxic agent, they undergo a series of changes necessary to transform them * To whom correspondence should be addressed. Fax: +3906- 49387140. E-mail: santini@iss.it. † Laboratorio di Ultrastrutture, Istituto Superiore di Sanita `. ‡ Istituto Nazionale per la Fisica della Materia, Unita ` di Napoli, Complesso Universitario Monte S. Angelo. § Dipartimento di Scienze Fisiche, Universita ` di Napoli “Federico II”, Complesso Universitario Monte S. Angelo. | Istituto di Chimica Biomolecolare del Consiglio Nazionale delle Ricerche, Comprensorio Olivetti. ⊥ UTS Biotecnologie, Protezione dell’Ambiente e degli Ecosistemi, ENEA. 63 Chem. Res. Toxicol. 2004, 17, 63-74 10.1021/tx034118f CCC: $27.50 © 2004 American Chemical Society Published on Web 12/19/2003