Table 1.
Percentages of nuclei in hypodiploid, diploid, hyperdiploid, tetraploid and hypertetraploid classes based on DNA content.
Figure 1.
Percentage of aneuploid nuclei after chrysotile treatment and recovery.
Nuclear DNA content of HK2 control and chrysotile (250 µg/ml, 125 µg/ml or 62.5 µg/ml) -treated cells (for 48 h) allowed recovering in fiber-free medium for 2, 4 or 8 days was quantified, and nuclei with DNA content >5.1 C were considered aneuploid. The percentages shown are background (the percentage of aneuploidy in control cells, 0.25%)-subtracted. Chrysotile treatment led to aneuploidy that persisted after 8 days of recovery (P<0.001).
Table 2.
Percentage of aneuploid nuclei after chrysotile exposure and recovery.
Table 3.
Percentages of control and chrysotile-treated HK2 cells in the different phases of the cell cycle.
Figure 2.
Chrysotile effects on the cell cycle and the percentage of cells in metaphase, anaphase and telophase in HK2 cells.
Cells treated with chrysotile and allowed to recover for 2, 4 or 8 days in fiber-free medium were analyzed by flow cytometry and by immunofluorescence. A) Histograms in linear (red) and log (colored) scales from flow cytometry show the effects of chysotile on the cell cycle, specifically an increase in the number of G2/M and hypertetraploid cells; B) chrysotile-treated cells recovered for 2 and 4 days show an increase in the number of cells in metaphase and a decrease in cells in anaphase and telophase compared with control cells (P<0.01 for 48 h and P<0.001 for 4 days).
Figure 3.
Alterations in the morphology of HK2 cells after 24 h or 48 h of chrysotile treatment.
Cells were processed by immunofluorescence to visualize nuclei, actin filaments and microtubules, and chrysotile fibers were observed by their autofluorescence. A) Confocal images of HK2 cells treated with chrysotile for 24 h or 48 h showing long, thick fibers interacting with the cells and multipolar mitosis; B) the alterations in cell morphology were analyzed, and after 24 h or 48 h of chrysotile treatment, the number of binucleated and multinucleated cells increased, as well the number of micronucleated cells, apoptotic cells and cells in multipolar mitosis (P<0.001).
Figure 4.
Alterations in the morphology of HK2 cells after 48 h of chrysotile treatment followed by 4 days and 8 days of recovery.
Cells were examined using immunofluorescence to visualize nuclei, actin filaments and microtubules, and chrysotile fibers were observed by their autofluorescence. A) Confocal images of control HK2 cells and cells treated with chrysotile for 48 h and allowed to recover for 2 days, 4 days or 8 days, showing cell morphology and the presence of chrysotile fibers. After 2 days of recovery, long fibers were observed to interact with the cells surface; however, after 4 and 8 days of recovery, only fiber fragments were observed; B) the alterations in cell morphology were quantified, and after 48 h of chrysotile treatment and 4 days of recovery, the number of bi/multinucleated cells and apoptotic cells decreased, but was still higher than controls (P = 0.30 for bi/multinucleated after 4 days and P = 0.05 for apoptotic cells). The number of micronucleated cells in the chrysotile-treated group remained greater that in the control group (P<0.001 after 4 days), and the number of cells in multipolar mitosis was greatest (P<0.001). After 8 days of recovery, the number of cells in multipolar mitosis cells remained higher than in control cells (P = 0.02).
Figure 5.
Alterations in VERO cell morphology after 48 h of chrysotile treatment and 24 h of recovery.
Cells were examined by using submitted to immunofluorescence to visualize the nuclei, actin filaments and microtubules, and chrysotile fibers were observed by their autofluorescence. A) Confocal images of control cells and cells treated with chrysotile for 48 h and allowed to recover for 24 h, showing cell morphology and the presence of chrysotile fibers. After recovery, long fibers were observed to interact with the cells, and multinucleated cells, micronucleated cells and cells in multipolar mitosis were observed; B) the alterations in cell morphology were quantified, and chrysotile treatment led to increased numbers of micronucleated cells, bi/multinucleated cells, apoptotic cells and cells in multipolar mitosis (P<0.001).
Figure 6.
Fates of HK2 cells in multipolar mitosis after chrysotile treatment.
Cells transfected with GFP-tagged α-tubulin were treated with chrysotile for 24 or 48 h and then observed by time-lapse spinning disk confocal microscopy. Cells in metaphase were observed for 2 to 3 h. A) A time series of maximal projection images showing a bipolar mitosis that generated only two daughter cells after 1 h in a control culture; B) a time series of maximal projection images showing a tripolar mitosis from a chrysotile-treated culture that did not progress through M phase; C) a time series of maximal projection images showing a chrysotile-treated cell that organized spindle poles in a tripolar fashion and progressed to anaphase and telophase generating three daughter cells; D) a time series of maximal projection images showing cytokinesis in a chrysotile-treated cell, generating three daughter cells that acquired interphase morphology; E) a time series of maximal projection images of a chrysotile-treated cell that entered anaphase generating four daughter cells; however, the cells merged during telophase and formed only two daughter cells.
Figure 7.
Alterations in interphase cells related to the formation of an abnormal number of centrosome.
Alterations that could be related to an abnormal number of centrosomes were observed in HK2 cells treated with chrysotile for 24 or 48 h. A) A time series of images showing a cell with two centrosome like-structures that did not progress to M phase as expected for a cell with two centrosomes; B) a time series of images showing two interphase cells linked by an intercellular bridge without a midbody organization. The cells approached each other during the period of observation, reflecting a regression of cytokinesis.