DNA Synthesis in Alveolar Macrophages and Other Changes in Lavaged Cells Following Exposure of CBA/H Mice to Cigarette Smoke

Abstract

Traditional methods to determine the proportion of cells in S-phase use radiolabeled precursors of DNA, such as 3 H-thymidine, which become incorporated into DNA during its synthesis and are visualized either in tissue sections or in cell preparations by autoradiography. At the Harwell Laboratory the effects of inhaled α-emitting actinides on the pulmonary alveolar macrophage population of the rodent lung are being studied. For this research the use of an autoradiographic technique to determine the proportion of cells in S-phase is inappropriate, because of the possible presence of competing sources of radioactivity in the cells under investigation. Consequently, an alternative method has been developed. In this method, 5-bromodeoxyuridine (BrdU), an analogue of thymidine, is incorporated into cells undergoing DNA synthesis. Fluorescein-conjugated monoclonal antibodies, highly specific for BrdU substituted DNA, are available commercially and may be used as a probe for BrdU-labeled cells. This technique for identifying cells in S-phase has been described previously for the flow cytometric analysis of cell suspensions and for cells in tissue sections. An adaptation of this technique for use on cytocentrifuge preparations of cells recovered from mouse lung by bronchoalveolar lavage has been developed and its use is described. Some preliminary results of a short-term experiment with CBA/H mice to determine the effects of exposure to cigarette smoke on the DNA synthesis of alveolar macrophages are also included.