Stem Cells, Vol. 17, No. 2, 125-126,
March 1999
© 1999 AlphaMed Press
Cotinine (a Metabolite of Nicotine) Suppresses the Growth of Hematopoietic Progenitor Cells at the Concentration Range Equivalent to Its Serum Levels in Smokers
Masuhiro Takahashi,
Naoya Shigeno,
Miwako Narita,
Hidenobu Takahashi,
Tatsuo Furukawa,
Tadashi Koike,
Yoshifusa Aizawa
Department of Medical Technology, College of Biomedical Technology, Niigata University, 2-746, Asahimachi, Niigata 951 Japan
First Department of Internal Medicine, School of Medicine, Niigata University, 1-754, Asahimachi, Niigata 951 Japan
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To the Editor:
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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 unit-granulocyte/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-GM- and 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 CFU-GM, 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 CFU-E 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).
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Figure 1. The effects of L-nicotine (A) and L-cotinine (B) on in vitro colony formation of CFU-GM, CFU-E, and BFU-E. Mean number of control colonies (triplicate) derived from CFU-GM, CFU-E and BFU-E were 59, 64, and 27 in 104 marrow mononuclear cells, respectively. Two additional experiments demonstrated similar results.
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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, although there is a tendency toward decreased erythrocyte counts, mean corpuscular hemoglobin and mean corpuscular volume are markedly increased [2]. On the contrary, CFU-E and BFU-E were suppressed by the addition of cotinine at the serum level concentrations. Although it has been reported that smokers' polycythemia is mainly caused by an increase in the serum erythropoietin level induced by oxygen deficiency [5], it is unlikely that smoking-associated macrocytosis without erythrocytosis is attributed merely to a stimulation of erythropoiesis by erythropoietin. Smokers' abnormalities of erythropoiesis are also considered to be synthesized by various factors, including the erythropoiesis-inhibitory activity of cotinine.
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References
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Takahashi M, Terao A, Murata M et al. Hematological abnormalities caused by smoking: the association with myelodysplastic syndrome. Niigata Med J 1997:111:647-655.
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Muranaka H, Tamada T, Higashi E et al. Microdetermination of nicotine and its metabolites in biological fluid. III. Effect of smoking on plasma nicotine, plasma cotinine and other biochemical variables in human being. Rinshokagaku 1981:10:127-135.
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Erslev AJ. Secondary polycythemia. In: Beutler E, Lichtman MA, Coller BS et al., eds. Williams Hematology. 5th Edition. New York: McGraw-Hill, 1995;714-726.
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