Mechanism of induction of binucleated cells by multiwalled carbon nanotubes as revealed by live-cell imaging analysis
© The Author(s) 2015
Received: 5 November 2014
Accepted: 17 February 2015
Published: 16 June 2015
Asbestos-induced formation of mesothelioma has been attributed to phenotypic and morphological changes in cells caused by polyploidization and aneuploidization, and multiwalled carbon nanotubes (MWCNTs) are suspected to have similar adverse effects due to the similarity in their physical form. MWCNTs and crocidolite, a kind of asbestos, show similar genotoxicity characteristics in vitro, including induction of binucleated cells. We here focused on the mechanisms underlying polyploidization during cell division on exposure to MWCNTs and conducted confocal live-cell imaging analysis using MDA-435 human breast cancer cells in which chromosomes and centromeres were visualized using fluorescent proteins.
During anaphase, relatively short MWCNT fibers (approximately 5 μm) migrated rapidly to either of the daughter cells, whereas some long MWCNT fibers (approximately 20 μm) remained inside the contractile ring and induced the formation of binucleated cells through impairment of cytokinesis. This toxicity mechanism has also been observed with crocidolite.
Our findings indicate that the mechanism of polyploidization by MWCNTs is very similar to that observed with crocidolite.
KeywordsPolyploidization Crocidolite Cytokinesis
Multiwalled carbon nanotubes (MWCNTs) have been suggested to be similar to crocidolite in terms of toxicity, given the similarity in their physical form [1–3]. Some animal studies have indicated that similar to crocidolite, intraperitoneally administered MWCNTs induce mesothelioma with a high frequency [4–6]. These results are generally consistent with the “Stanton–Pott hypothesis” that asbestos fibers with a diameter of ≤0.25 μm and length of ≥20 μm are highly carcinogenic [7, 8]. Muller et al. reported no increase in carcinogenesis in Wistar rats following intraperitoneal administration of short carbon nanotube (CNT) fibers whose length was less than that indicated in the hypothesis (average length: ≤1 μm) . Only a limited number of in vivo studies have investigated the genotoxicity of MWCNTs [10, 11]. Kato et al.  performed intratracheal injection of MWCNTs (width: 70–110 nm, length: 1–4 μm) in wild-type ICR mice and found that the results of a comet assay, oxidative DNA adduct assay, and immunohistochemical analysis of nitric oxide synthase with the lung tissue were all positive. Therefore, the genotoxicity of MWCNTs has been shown to result predominantly from oxidative stress induced by excessive inflammatory responses to CNT fibers.
MWCNTs and asbestos show similar genotoxicity characteristics even in cell culture experiments, and both are known to induce polyploid cells (and multinucleated cells) with a high frequency [12–15]. Chromosomal abnormalities caused by polyploidization and aneuploidization alter the expression of a variety of genes involved in carcinogenesis and thus are believed to be closely related to asbestos-induced mesothelioma and bronchial cancer, as observed in animal studies [16, 17]. Jensen et al. conducted time-lapse analysis using a microscope applicable for live-cell observation to determine the mechanisms underlying the induction of abnormal binucleated and multinucleated cells by asbestos . They observed that comparatively long crocidolite (15–50 μm) fibers were trapped in the contractile ring during anaphase in LLC-MK2 epithelial cells, which created a physical barrier to cytokinesis, eventually causing formation of binucleated cells. On the other hand, many reports have demonstrated a causal role of MWCNTs in cell multinucleation and polyploidization; however, only few have directly demonstrated the mechanism underlying the occurrence of these aberrations .
In this study, we conducted time-lapse analysis with a high-resolution confocal live-cell imaging system to elucidate the mechanism involved in the MWCNT-induced formation of binucleated cells using dichromatically visualized human cells in which chromosomes and centromeres were stained with different fluorescent proteins. We found that short CNT fibers (approximately 5 μm) migrated to either of the daughter cells immediately after chromosome segregation, whereas long fibers (approximately 20 μm) formed a bridge structure between the 2 daughter cells during anaphase and induced the formation of binucleated cells by impeding cytokinesis. This physical disruption of cytokinesis was very similar to the asbestos-induced disruption described above.
Materials and methods
The MWCNTs used in this study were MWCNT-7 (Lot No.060125-01k) manufactured by Mitsui & Co., Ltd. (Ibaraki, Japan), which was same batch used in the study reported by Takagi et al. . According to the report, these MWCNT fibers were approximately 100 nm in diameter and contained 27.5 % of MWCNTs ≥5 μm in length. The MWCNTs were suspended in 100 % fetal bovine serum (Gibco, Invitrogen, NY, USA) at a concentration of 1 mg/mL and were autoclaved for 15 min at 121 °C. Thereafter, Tween 80 (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was added to a final concentration of 1.0 % in fetal bovine serum. The resulting mixture was subjected to ultrasonication using an ultrasonic homogenizer (VP30s, TAITEC Co., Saitama, Japan).
Dichromatically visualized MDA-435 human breast cancer cells, in which chromosomes and centromeres were stained with a red fluorescent protein (mCherry–Histone H3 fusion) and green fluorescent protein (EGFP–CENP-A fusion), respectively, were kindly provided by Dr. Kenji Sugimoto (Osaka Prefecture University, Osaka, Japan) . The cells were cultured at 37 °C (5 % CO2, 100 % humidity) in Dulbecco’s Modified Eagle’s medium (DMEM) (Nacalai Tesque, Kyoto, Japan), supplemented with 10 % fetal bovine serum. MDA-435 cell line, isolated from ductal adenocarcinoma of female breast, is aneuploid with most chromosome counts in the 55–60 range (modal number = 56) .
We used an FV1000 laser fluorescence microscope (Olympus Corp., Tokyo, Japan) equipped with a humid chamber to capture images as the cells were cultured. We also used a multi-Ar and He–Ne G laser and an objective lens with 60× magnification (1.20 Numerical Aperture). For imaging, 5 × 105 MDA-435 cells were cultured in 2 mL of DMEM containing 10 % fetal bovine serum (37 °C, 5 % CO2, 100 % humidity) in a 35-mm glass base dish (IWAKI, ASAHI GLASS CO., Ltd., Tokyo, Japan). To minimize cytotoxicity of the laser, we conducted the experiments at a weak laser output such that ≥50 % cells divided after 24 h among the control cells. The acquired images were edited using Volocity Software (PerkinElmer Inc., Massachusetts, USA), and the resulting moving images were analyzed. When MWCNTs were added to the medium (final concentration: 0, 12.7, 25.3, or 50.6 μg/mL), images of a visual field containing a large number of cells in metaphase were taken at 5-min intervals for a period of 48–72 h (in the z-axial direction, photographs were taken every 2.0 μm). All cells in the visual field were counted for each MWCNT concentration, and the incidences (%) of cells that completed cell division, cells that were unable to undergo cell division and subsequently died, and cells that became binucleated were calculated by dividing the number of such cells by the total cell count. We did not take statistical analysis for the incidences of divided, dead, and bi-nucleated cells, since the images of a visual field containing a large number of cells in metaphase were intentionally selected. Bi- and multi-nucleated cells had more than two nuclei in a cell. The cell death was defined as mitotic catastrophe during M-phase (from prophase to telophase). Approximate length of MWCNT fiber was estimated from bar length given in each images.
Results and discussion
Endocytosis of MWCNTs
Cytotoxicity of MWCNTs and the incidence of binucleated cells
Observation of cell division involving MWCNTs using live-cell imaginga
Total of recorded cells
No. of divided cells
No. of dead cells during mitosis
No. of binucleated cells during mitosis
93 (40 %)
4 (1.7 %)
40 (38 %)
4 (3.2 %)
49 (27 %)
11 (5.9 %)
5 (3.0 %)
34 (12 %)
21 (7.6 %)
1 (0.4 %)
Formation of binucleated cells through disturbance of cytokinesis
MWCNTs used in this experiment contained approximately 3500 ppm of iron and thus may have caused oxidative DNA damage to the cell genome by the reactive oxygen species formed during the Fenton reaction [4, 23, 24]. Nonetheless, even with the analysis system used in this study capable of detecting extremely small micronuclei , we did not observe any abnormality (such as micronuclei formation or abnormal multipolar division involving multiple centromeres) attributable to incubation with CNT fibers within the 72-h imaging period. Asakura et al. used MWCNT-7 (iron content: 4400 ppm) obtained from the same manufacturer to perform a chromosome abnormality test, an in vitro micronucleus test, and the Hprt gene mutation assay in Chinese hamster lung (CHL/IU) cells. The test results were all negative, but they observed an increase in the number of binucleated and polyploid cells with an increase in MWCNT concentration . These results are consistent with the mechanism of induction of binucleated cells observed in this study and suggest that CNT fibers containing comparatively less iron result in greater physical disruption of cytokinesis than DNA damage by reactive oxygen species. In other words, MWCNTs may allow karyokinesis to proceed and may induce abnormal cells that are either binuclear or tetranuclear, considering that MWCNTs inhibit cytokinesis but do not cause lethal damage to the nucleus or chromosomes to the extent that prevents cell division (Fig. 1).
In conclusion, we observed that comparatively long MWCNTs (approximately ≥20 μm) inhibited cytokinesis during cell division and induced the formation of binucleated cells, whereas short MWCNTs did not. These results indicate that the mechanism of induction of binucleated cells by MWCNTs is very similar to that observed with crocidolite.
We thank Dr. Kenji Sugimoto for the generous gift of dichromatically visualized MDA-435 cells. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare (H24-Food-General-011 and H24-kagaku-shitei-009).
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