Cotinine (a Metabolite of Nicotine) Suppresses the Growth of Hematopoietic Progenitor Cells at the Concentration Range Equivalent to Its Serum Levels in Smokers

Smoking is associated with various hematological abnormalities such as leukocytosis and increased hemoglobin concentration and hematocrit [1]. Our previous study showed that macrocytosis and leukocytosis were prominent in smokers [2]. On the other hand, mainstream cigarette smoke components are divided into particle-phase components and gas-phase components. Nicotine is a predominant component of the particle phase and possesses a high biological activity [3]. In order to clarify the effects of nicotine and its metabolite, cotinine, on hematopoiesis, in vitro colony assay of hematopoietic progenitor cells with the addition of nicotine or cotinine was performed. Bone marrow cells were aspirated in a heparinized syringe from healthy volunteers. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation. L-nicotine and L-cotinine were purchased from Sigma (St. Louis, MO). Colony formations of colony-forming unitgranulocyte/macrophage (CFU-GM), BFU-E, and CFU-erythroid (CFU-E) were performed in a 35-mm culture dish containing 104 marrow mononuclear cells and MethoCult GF + H4435 (Stem Cell Technologies; Vancouver, BC). CFU-GMand BFU-E-derived colonies were counted after culturing for 14 days on an inverted microscope, and CFU-E-derived colonies were counted after seven days. L-nicotine or L-cotinine were added to the cultures at various concentrations during the whole culture period because hematopoietic progenitor cells are repeatedly exposed to nicotine and cotinine in vivo. L-nicotine did not suppress the colony formations of CFUGM, BFU-E, and CFU-E at the concentration range equivalent to its serum level in smokers, which is 2.8-32.99 ng/ml [4]. In the higher concentration of nicotine, the colony formation was moderately suppressed (Fig. 1). L-cotinine, however, suppressed the colony formations of CFU-GM, BFU-E, and CFUE in a dose-dependent fashion at the concentration range equivalent to its serum level in smokers, which is 21.9-543.5 ng/ml [4]. At a higher concentration of cotinine, the colony formation was markedly suppressed. The 50% inhibitory concentration (IC50) of L-cotinine was presumed to be 250-500 ng/ml for BFU-E and CFU-E, respectively, and 100-200 ng/ml for CFU-GM (Fig. 1). The present in vitro colony assay demonstrated that cotinine, but not nicotine, suppressed the colony formation of CFU-GM, BFU-E, and CFU-E in a dose-dependent fashion at the concentration range equivalent to its serum level in smokers. Our previous comparative analysis of hematological data between nonsmokers and smokers demonstrated significant increases of leukocyte counts, mean corpuscular hemoglobin, and mean corpuscular volume in smokers [2]. The in vitro suppression of granulopoiesis by cotinine is against the leukocytosis observed in smokers. Since the concentration of cotinine, which suppressed CFU-GM-derived colony growth, was within the serum levels of cotinine in smokers, the suppression of granulopoiesis due to cotinine is considered to be present in vivo. However, nicotine/cotinine is not the only component of smoke, and there seem to be various factors associated with stimulation of granulopoiesis in smokers, such as occult chronic bronchitis or catecholamine-associated change in distribution of leukocytes from a marginal pool to a circulating pool. As smoke contains innumerable components having biological activity, there may be several components in smoke which increase leukocyte counts. Smokers’ leukocytosis seems to be due to a composite of various factors, including cotinine suppression of granulopoiesis. In smokers,