Formaldehyde induces micronuclei in mouse erythropoietic cells and suppresses the expansion of human erythroid progenitor cells

Abstract

Although formaldehyde (FA) has been classified as a human leukemogen, the mechanisms of leukemogenesis remain elusive. Previously, using colony-forming assays in semi-solid media, we showed that FA exposure in vivo and in vitro was toxic to human hematopoietic stem/progenitor cells. In the present study, we have applied new liquid in vitro erythroid expansion systems to further investigate the toxic effects of FA (0-150 μM) on cultured mouse and human hematopoietic stem/progenitor cells. We determined micronucleus (MN) levels in polychromatic erythrocytes (PCEs) differentiated from mouse bone marrow. We measured cell growth, cell cycle distribution, and chromosomal instability, in erythroid progenitor cells (EPCs) expanded from human peripheral blood mononuclear cells. FA significantly induced MN in mouse PCEs and suppressed human EPC expansion in a dose-dependent manner, compared with untreated controls. In the expanded human EPCs, FA slightly increased the proportion of cells in G2/M at 100 μM and aneuploidy frequency in chromosomes 7 and 8 at 50 μM. Our findings provide further evidence of the toxicity of FA to hematopoietic stem/progenitor cells and support the biological plausibility of FA-induced leukemogenesis.

Keywords: Aneuploidy; Erythroid progenitor; Formaldehyde; Micronuclei.

Figure 1. MN frequency in mouse erythropoietic cells after FA treatment

Three independent experiments were conducted. Data is presented as mean ± standard error (SE). ^**** represents P < 0.0001, compared to the untreated control and P_trend represents the p-value of the dose-response trend test. MN frequencies were significantly increased at each dose of FA and across all doses in a dose-dependent manner.

Figure 2. Human EPC expansion from peripheral blood

Three experiments on three blood donors were conducted. A. Ratio of EPCs relative to day 0 during the course of the culture. Expansion began at day 6 and increased by ~12-fold by day 10. B. Expression levels (%) of erythroid markers after expansion (day 10). EPCs expanded ~100-fold by day 10. Data is presented as mean ± standard error (SE).

Figure 3. Human EPC expansion after FA treatment

Six experiments on five blood donors were conducted. A. Ratio of EPCs relative to vehicle control on day 10. ^*** and ^**** represent P < 0.001 and P < 0.0001, respectively, compared to the untreated control and P_trend represents the p-value of the dose-response trend test. The number of EPCs generated as a percentage of untreated control were significantly reduced by 100 and 150 µM FA and across the dose-range in a dose-dependent manner. B. Expression levels (%) of erythroid markers after expansion (day 10). Levels were unchanged by FA. Data is presented as mean ± SE.

Figure 4. Cell cycle distribution of the expanded human EPCs after FA treatment

Four experiments on three blood donors were conducted. Data is presented as mean ± SE. A. Cell cycle distribution. At 100 µM FA, the percentage of cells in G0 and G1 phase was significantly decreased (44.1%) compared to that of the untreated control (48.9%, P < 0.05) and the percentage of cells in G2 and M phase was significantly increased (21.5%) compared to that of the untreated control (17.9%, P < 0.05). The proportion of cells in S phase was not significantly changed by FA treatment. B. Proliferation index. The index was significantly increased at 100 µM FA (0.54) compared to the untreated control (0.49, P < 0.05). ^* represents P < 0.05, compared to the untreated control and P_trend represents the p-value of the dose-response trend test.