Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Mechanism of induction of binucleated cells by multiwalled carbon nanotubes as revealed by live-cell imaging analysis

Mechanism of induction of binucleated cells by multiwalled carbon nanotubes as revealed by... Introduction: 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. Findings: 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. Conclusions: Our findings indicate that the mechanism of polyploidization by MWCNTs is very similar to that observed with crocidolite. Keywords: Polyploidization, Crocidolite, Cytokinesis Introduction performed intratracheal injection of MWCNTs (width: 70– Multiwalled carbon nanotubes (MWCNTs) have been 110 nm, length: 1–4 μm) in wild-type ICR mice and found suggested to be similar to crocidolite in terms of toxicity, that the results of a comet assay, oxidative DNA adduct given the similarity in their physical form [1–3]. Some assay, and immunohistochemical analysis of nitric oxide animal studies have indicated that similar to crocidolite, synthase with the lung tissue were all positive. Therefore, intraperitoneally administered MWCNTs induce meso- the genotoxicity of MWCNTs has been shown to result pre- thelioma with a high frequency [4–6]. These results are dominantly from oxidative stress induced by excessive in- generally consistent with the “Stanton–Pott hypothesis” flammatory responses to CNT fibers. that asbestos fibers with a diameter of ≤0.25 μmand MWCNTs and asbestos show similar genotoxicity char- length of ≥20 μm are highly carcinogenic [7, 8]. Muller acteristics even in cell culture experiments, and both are et al. reported no increase in carcinogenesis in Wistar rats known to induce polyploid cells (and multinucleated cells) following intraperitoneal administration of short carbon with a high frequency [12–15]. Chromosomal abnormal- nanotube (CNT) fibers whose length was less than that in- ities caused by polyploidization and aneuploidization alter dicated in the hypothesis (average length: ≤1 μm) [9]. Only the expression of a variety of genes involved in carcinogen- a limited number of in vivo studies have investigated the esis and thus are believed to be closely related to asbestos- genotoxicity of MWCNTs [10, 11]. Kato et al. [11] induced mesothelioma and bronchial cancer, as observed in animal studies [16, 17]. Jensen et al. conducted time- * Correspondence: m-yasui@nihs.go.jp lapse analysis using a microscope applicable for live-cell Division of Genetics and Mutagenesis, National Institute of Health Sciences, observation to determine the mechanisms underlying the 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan induction of abnormal binucleated and multinucleated Full list of author information is available at the end of the article © 2015 Yasui et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yasui et al. Genes and Environment (2015) 37:6 Page 2 of 6 cells by asbestos [18]. They observed that comparatively Live-cell imaging long crocidolite (15–50 μm) fibers were trapped in the We used an FV1000 laser fluorescence microscope contractile ring during anaphase in LLC-MK epithelial (Olympus Corp., Tokyo, Japan) equipped with a humid cells, which created a physical barrier to cytokinesis, even- chamber to capture images as the cells were cultured. tually causing formation of binucleated cells. On the other We also used a multi-Ar and He–Ne G laser and an ob- hand, many reports have demonstrated a causal role of jective lens with 60× magnification (1.20 Numerical MWCNTs in cell multinucleation and polyploidization; Aperture). For imaging, 5 × 10 MDA-435 cells were cul- however, only few have directly demonstrated the mechan- tured in 2 mL of DMEM containing 10 % fetal bovine ism underlying the occurrence of these aberrations [19]. serum (37 °C, 5 % CO , 100 % humidity) in a 35-mm In this study, we conducted time-lapse analysis with a glass base dish (IWAKI, ASAHI GLASS CO., Ltd., high-resolution confocal live-cell imaging system to elu- Tokyo, Japan). To minimize cytotoxicity of the laser, we cidate the mechanism involved in the MWCNT-induced conducted the experiments at a weak laser output such formation of binucleated cells using dichromatically vi- that ≥50 % cells divided after 24 h among the control sualized human cells in which chromosomes and centro- cells. The acquired images were edited using Volocity meres were stained with different fluorescent proteins. Software (PerkinElmer Inc., Massachusetts, USA), and We found that short CNT fibers (approximately 5 μm) the resulting moving images were analyzed. When migrated to either of the daughter cells immediately MWCNTs were added to the medium (final concentra- after chromosome segregation, whereas long fibers (ap- tion: 0, 12.7, 25.3, or 50.6 μg/mL), images of a visual proximately 20 μm) formed a bridge structure between field containing a large number of cells in metaphase the 2 daughter cells during anaphase and induced the were taken at 5-min intervals for a period of 48–72 h (in formation of binucleated cells by impeding cytokinesis. the z-axial direction, photographs were taken every This physical disruption of cytokinesis was very similar 2.0 μm). All cells in the visual field were counted for to the asbestos-induced disruption described above. each MWCNT concentration, and the incidences (%) of cells that completed cell division, cells that were unable Materials and methods to undergo cell division and subsequently died, and cells MWCNTs that became binucleated were calculated by dividing the The MWCNTs used in this study were MWCNT-7 (Lot number of such cells by the total cell count. We did not No.060125-01k) manufactured by Mitsui & Co., Ltd. take statistical analysis for the incidences of divided, (Ibaraki, Japan), which was same batch used in the study dead, and bi-nucleated cells, since the images of a visual reported by Takagi et al. [4]. According to the report, field containing a large number of cells in metaphase these MWCNT fibers were approximately 100 nm in were intentionally selected. Bi- and multi-nucleated cells diameter and contained 27.5 % of MWCNTs ≥5 μmin had more than two nuclei in a cell. The cell death was length. The MWCNTs were suspended in 100 % fetal defined as mitotic catastrophe during M-phase (from bovine serum (Gibco, Invitrogen, NY, USA) at a concen- prophase to telophase). Approximate length of MWCNT tration of 1 mg/mL and were autoclaved for 15 min at fiber was estimated from bar length given in each 121 °C. Thereafter, Tween 80 (Tokyo Chemical Industry images. Co., Ltd., Tokyo, Japan) was added to a final concentra- tion of 1.0 % in fetal bovine serum. The resulting mix- Results and discussion ture was subjected to ultrasonication using an ultrasonic Endocytosis of MWCNTs homogenizer (VP30s, TAITEC Co., Saitama, Japan). We conducted time-lapse imaging at the MWCNT con- centration of 50.6 μg/mL to determine how MWCNTs Cell culture undergo endocytosis. The results showed that some cells Dichromatically visualized MDA-435 human breast cancer actively ingested and incorporated CNT fibers within a cells, in which chromosomes and centromeres were stained few hours after addition of MWCNTs (Additional file 1, with a red fluorescent protein (mCherry–Histone H3 the right side in the movie) and other cells did not. Simi- fusion) and green fluorescent protein (EGFP–CENP-A fu- lar findings have been reported in a previous study in sion), respectively,werekindlyprovidedbyDr. KenjiSugi- which MWCNTs were easily incorporated within 24 h in moto (Osaka Prefecture University, Osaka, Japan) [20]. The experiments with human neonatal epidermal keratino- cells were cultured at 37 °C (5 % CO , 100 % humidity) in cytes [22]. CNT fibers that were incorporated into a cell Dulbecco’s Modified Eagle’s medium (DMEM) (Nacalai remained there for a long time without being ejected Tesque,Kyoto,Japan),supplemented with 10 % fetal bovine and migrated to either of the daughter cells after cell serum. MDA-435 cell line, isolated from ductal adenocar- division. In addition, we observed that CNT fibers were cinoma of female breast, is aneuploid with most chromo- frequently stuck to the cell surface. After 72 h, virtually some counts in the 55–60 range (modal number = 56) [21]. no abnormal cells (such as multinucleated cells) were Yasui et al. Genes and Environment (2015) 37:6 Page 3 of 6 Control CNT treatment CNT only Fig. 1 Comparison of untreated (control) and MWCNT (50.6 μg/mL) treated cells after 72 h. Many abnormal cells (such as multinucleated cells) were found in the MWCNT-treated cell group (the white dotted line in the image). Bar, 41 μm found in the untreated (control) group of cells (Fig. 1). 40 (38 %), 49 (27 %), and 34 (12 %) cells completed cell On the other hand, in the MWCNT-treated cell group, division and 4 (3.2 %), 11 (5.9 %), and 21 (7.6 %) cells most cells were entangled with CNT fibers, and many died at concentrations of 12.7, 25.3, and 50.6 μg/mL, multinucleated cells were observed (Fig. 1, the white respectively. Thus, the number of cells that completed dotted line). cell division decreased and the number of dead cells increased with an increase in MWCNT concentration, Cytotoxicity of MWCNTs and the incidence of binucleated indicating the concentration-dependent cytotoxicity of cells MWCNTs. However, endocytosis of MWCNT was dif- Time-lapse images of MDA-435 cells in the medium ferent depending on individual cells, as described above. containing MWCNTs (0, 12.7, 25.3, or 50.6 μg/mL) were Some cells did not undergo cell death when incor- taken up to 72 h. The experiment was conducted 3 porated a small number of CNT fibers. Actually, both times with each concentration, and 232, 112, 170, and incidences of divided cells between control and low 282 cells were imaged in the visual field, respectively concentration (12.7 μg/mL) were almost same (40 and (Table 1). Among the untreated cells, 93 (40 %) of the 38 %), as shown in Table 1. When ingested many CNT 232 cells completed cell division and only 4 (1.7 %) cells fibers even at low concentration, the cells gave rise to died during cell division. In the MWCNT-treated group, mitotic catastrophe. In addition, the number of Table 1 Observation of cell division involving MWCNTs using live-cell imaging MWCNTs (μg/mL) Experiment Total of recorded cells No. of divided cells No. of dead cells during mitosis No. of binucleated cells during mitosis 0 1 76 37 1 0 274 27 2 0 382 29 1 0 Total 232 93 (40 %) 4 (1.7 %) 0 12.7 1 33 16 0 0 232 13 1 0 347 11 3 0 Total 112 40 (38 %) 4 (3.2 %) 0 25.3 1 38 5 2 1 251 19 2 2 381 25 7 2 Total 170 49 (27 %) 11 (5.9 %) 5 (3.0 %) 50.6 1 86 12 10 1 2 101 11 5 0 395 11 6 0 Total 282 34 (12 %) 21 (7.6 %) 1 (0.4 %) Statistical analysis for the incidences of divided, dead, and binucleated cells was not done, as described in Materials and Methods Yasui et al. Genes and Environment (2015) 37:6 Page 4 of 6 binucleated cells was 0 (0 %), 5 (3 %), and 1 (0.4 %) at (the white arrow in Fig. 2a) and presumably exerted no the respective concentrations. A possible reason for the lethal damage during karyokinesis or cytokinesis. In lower incidence of binucleated cells at the highest dose contrast, cell division involving long CNT fibers (ap- of 50.6 μg/mL than at the lower dose of 25.3 μg/mL was proximately 20 μm) took almost 3 h (Fig. 2b). The long that the cells underwent cell division less frequently at CNT fibers formed a bridge between the 2 daughter the highest dose, as described above; furthermore, a very cells during anaphase (arrowheads in Fig. 2b), and large number of cells did not enter the mitotic phase remained in the contractile ring (2:30). Thereafter, and remained in interphase during imaging. karyokinesis was only slightly delayed and was com- pleted normally without micronuclei formation (2:50). Formation of binucleated cells through disturbance of However, approximately at the same time, cytokinesis cytokinesis was impeded by the CNT fiber bridge; consequently, the Time-lapse images of typical cell division involving constriction of the contractile ring was gradually MWCNTs are shown in Fig. 2. Normal cell division was abrogated (3:20). Thereafter, the borderline between the completed within 30 min of chromosome segregation 2 cells disappeared (4:40), resulting in the formation of during metaphase. Cell division involving short CNT fibers binucleated cells (5:40, the white dotted line). This dis- (approximately 5 μm) (Fig. 2a) occurred smoothly, similar ruption of cytokinesis was very similar to the process to that in the untreated group of cells, and was completed observed with crocidolite [18]. within 30 min. These short CNT fibers migrated to the MWCNTs used in this experiment contained approxi- daughter cells immediately after chromosome segregation mately 3500 ppm of iron and thus may have caused Fig. 2 Time-lapse analysis of cell division involving MWCNT fibers (25.3 μg/mL). a Short MWCNT fibers (approximately 5 μm) did not inhibit cell division. b Long MWCNT fibers (approximately 20 μm) inhibited cytokinesis and induced the formation of binucleated cells (the white dotted line in the image at 5 h 40 min). Time (h:min) is shown at the top. Bar, 41 μm Yasui et al. Genes and Environment (2015) 37:6 Page 5 of 6 oxidative DNA damage to the cell genome by the react- Pharmaceutical Sciences, Teikyo Heisei University, 4-21-2 Nakano, Nakano-ku, Tokyo 164-8530, Japan. ive oxygen species formed during the Fenton reaction [4, 23, 24]. Nonetheless, even with the analysis system Received: 5 November 2014 Accepted: 17 February 2015 used in this study capable of detecting extremely small micronuclei [25], we did not observe any abnormality (such as micronuclei formation or abnormal multipolar References 1. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, division involving multiple centromeres) attributable to et al. Carbon nanotubes introduced into the abdominal cavity of mice incubation with CNT fibers within the 72-h imaging show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. period. Asakura et al. used MWCNT-7 (iron content: 2008;3:423–8. 2. Toyokuni S. Genotoxicity and carcinogenicity risk of carbon nanotubes. 4400 ppm) obtained from the same manufacturer to Adv Drug Deliv Rev. 2013;65:2098–110. perform a chromosome abnormality test, an in vitro 3. Donaldson K, Poland CA, Murphy FA, MacFarlane M, Chernova T, Schinwald micronucleus test, and the Hprt gene mutation assay in A. Pulmonary toxicity of carbon nanotubes and asbestos - similarities and differences. Adv Drug Deliv Rev. 2013;65:2078–86. Chinese hamster lung (CHL/IU) cells. The test results 4. Takagi A, Hirose A, Nishimura T, Fukumori N, Ogata A, Ohashi N, et al. were all negative, but they observed an increase in the Induction of mesothelioma in p53+/− mouse by intraperitoneal application number of binucleated and polyploid cells with an of multi-wall carbon nanotube. J Toxicol Sci. 2008;33:105–16. 5. Takagi A, Hirose A, Futakuchi M, Tsuda H, Kanno J. Dose-dependent increase in MWCNT concentration [14]. These results mesothelioma induction by intraperitoneal administration of multi-wall are consistent with the mechanism of induction of binu- carbon nanotubes in p53 heterozygous mice. Cancer Sci. 2012;103:1440–4. cleated cells observed in this study and suggest that CNT 6. Sakamoto Y, Nakae D, Fukumori N, Tayama K, Maekawa A, Imai K, et al. Induction of mesothelioma by a single intrascrotal administration of fibers containing comparatively less iron result in greater multi-wall carbon nanotube in intact male Fischer 344 rats. J Toxicol Sci. physical disruption of cytokinesis than DNA damage by 2009;34:65–76. reactive oxygen species. In other words, MWCNTs may 7. Stanton MF, Layard M, Tegeris A, Miller E, May M, Morgan E, et al. Relation of particle dimension to carcinogenicity in amphibole asbestoses and other allow karyokinesis to proceed and may induce abnormal fibrous minerals. J Natl Cancer Inst. 1981;67:965–75. cells that are either binuclear or tetranuclear, considering 8. Pott F, Schlipkoter HW, Ziem U, Spurny K, Huth F. New results from implantation that MWCNTs inhibit cytokinesis but do not cause experiments with mineral fibres. In: Proceedings of a WHO/IARC Conference: Biological effects of man-made mineral fibres. Copenhagen: World Heath lethal damage to the nucleus or chromosomes to the Organization; 1984. p. 286–302. extent that prevents cell division (Fig. 1). 9. Muller J, Delos M, Panin N, Rabolli V, Huaux F, Lison D. Absence of In conclusion, we observed that comparatively long carcinogenic response to multiwall carbon nanotubes in a 2-year bioassay in the peritoneal cavity of the rat. Toxicol Sci. 2009;110:442–8. MWCNTs (approximately ≥20 μm) inhibited cytokinesis 10. Kim JS, Sung JH, Choi BG, Ryu HY, Song KS, Shin JH, et al. In vivo during cell division and induced the formation of genotoxicity evaluation of lung cells from Fischer 344 rats following 28 days binucleated cells, whereas short MWCNTs did not. of inhalation exposure to MWCNTs, plus 28 days and 90 days post-exposure. Inhal Toxicol. 2014;26:222–34. These results indicate that the mechanism of induction 11. Kato T, Totsuka Y, Ishino K, Matsumoto Y, Tada Y, Nakae D, et al. of binucleated cells by MWCNTs is very similar to that Genotoxicity of multi-walled carbon nanotubes in both in vitro and in vivo observed with crocidolite. assay systems. Nanotoxicology. 2013;7:452–61. 12. Jaurand MC, Bastie-Sigeac I, Renier A, Bignon J. Comparative toxicities of different forms of asbestos on rat pleural mesothelial cells. Environ Health Additional file Perspect. 1983;51:153–8. 13. Kenne K, Ljungquist S, Ringertz NR. Effects of asbestos fibers on cell division, cell survival, and formation of thioguanine-resistant mutants in Chinese Additional file 1: Time-lapse of endocytosis of cells exposed to hamster ovary cells. Environ Res. 1986;39:448–64. MWCNT (50.6 μg/mL). MWCNTs were easily incorporated within a few 14. Asakura M, Sasaki T, Sugiyama T, Takaya M, Koda S, Nagano K, et al. hours. CNT fibers that were incorporated into a cell remained there for a Genotoxicity and cytotoxicity of multi-wall carbon nanotubes in cultured long time without being ejected and migrated to either of the daughter Chinese hamster lung cells in comparison with chrysotile A fibers. J Occup cells after cell division. Bar, 41 μm. Health. 2010;52:155–66. 15. Muller J, Decordier I, Hoet PH, Lombaert N, Thomassen L, Huaux F, et al. Competing interests Clastogenic and aneugenic effects of multi-wall carbon nanotubes in The authors declare that they have no competing interest. epithelial cells. Carcinogenesis. 2008;29:427–33. 16. Oshimura M, Hesterberg TW, Tsutsui T, Barrett JC. Correlation of asbestos-induced Authors’ contributions cytogenetic effects with cell transformation of Syrian hamster embryo cells in MY and NK collected the data. TN prepared MWCNT samples. MY and MH culture. Cancer Res. 1984;44:5017–22. designed and critically discussed the study. All authors read and approved 17. Craighead JE, Akley NJ, Gould LB, Libbus BL. Characteristics of tumors and the final manuscript. tumor cells cultured from experimental asbestos-induced mesotheliomas in rats. Am J Pathol. 1987;129:448–62. Acknowledgments 18. Jensen CG, Jensen LC, Rieder CL, Cole RW, Ault JG. Long crocidolite We thank Dr. Kenji Sugimoto for the generous gift of dichromatically asbestos fibers cause polyploidy by sterically blocking cytokinesis. visualized MDA-435 cells. This study was supported by a Grant-in-Aid for Carcinogenesis. 1996;17:2013–21. Scientific Research from the Ministry of Health, Labour and Welfare 19. Sargent LM, Reynolds SH, Castranova V. Potential pulmonary effects of (H24-Food-General-011 and H24-kagaku-shitei-009). engineered carbon nanotubes: in vitro genotoxic effects. Nanotoxicology. 2010;4:396–408. Author details 20. Sugimoto K, Senda-Murata K, Oka S. Construction of three quadruple-fluorescent Division of Genetics and Mutagenesis, National Institute of Health Sciences, MDA435 cell lines that enable monitoring of the whole chromosome 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Faculty of segregation process in the living state. Mutat Res. 2008;657:56–62. Yasui et al. Genes and Environment (2015) 37:6 Page 6 of 6 21. Phillips KK, Welch DR, Miele ME, Lee JH, Wei LL, Weissman BE. Suppression of MDA-MB-435 breast carcinoma cell metastasis following the introduction of human chromosome 11. Cancer Res. 1996;56:1222–7. 22. Monteiro-Riviere NA, Nemanich RJ, Inman AO, Wang YY, Riviere JE. Multi-walled carbon nanotube interactions with human epidermal keratinocytes. Toxicol Lett. 2005;155:377–84. 23. Tabet L, Bussy C, Amara N, Setyan A, Grodet A, Rossi MJ, et al. Adverse effects of industrial multiwalled carbon nanotubes on human pulmonary cells. J Toxicol Environ Health A. 2009;72:60–73. 24. Kagan VE, Tyurina YY, Tyurin VA, Konduru NV, Potapovich AI, Osipov AN, et al. Direct and indirect effects of single walled carbon nanotubes on RAW 264.7 macrophages: role of iron. Toxicol Lett. 2006;165:88–100. 25. Yasui M, Koyama N, Koizumi T, Senda-Murata K, Takashima Y, Hayashi M, et al. Live cell imaging of micronucleus formation and development. Mutat Res. 2010;692:12–8. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genes and Environment Springer Journals

Mechanism of induction of binucleated cells by multiwalled carbon nanotubes as revealed by live-cell imaging analysis

Download PDF
6 pages

Loading next page...
 
/lp/springer-journals/mechanism-of-induction-of-binucleated-cells-by-multiwalled-carbon-n23N0xhMFE

References (26)

Publisher
Springer Journals
Copyright
Copyright © 2015 by The Author(s)
Subject
Biomedicine; Human Genetics
eISSN
1880-7062
DOI
10.1186/s41021-015-0003-y
pmid
27350803
Publisher site
See Article on Publisher Site

Abstract

Introduction: 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. Findings: 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. Conclusions: Our findings indicate that the mechanism of polyploidization by MWCNTs is very similar to that observed with crocidolite. Keywords: Polyploidization, Crocidolite, Cytokinesis Introduction performed intratracheal injection of MWCNTs (width: 70– Multiwalled carbon nanotubes (MWCNTs) have been 110 nm, length: 1–4 μm) in wild-type ICR mice and found suggested to be similar to crocidolite in terms of toxicity, that the results of a comet assay, oxidative DNA adduct given the similarity in their physical form [1–3]. Some assay, and immunohistochemical analysis of nitric oxide animal studies have indicated that similar to crocidolite, synthase with the lung tissue were all positive. Therefore, intraperitoneally administered MWCNTs induce meso- the genotoxicity of MWCNTs has been shown to result pre- thelioma with a high frequency [4–6]. These results are dominantly from oxidative stress induced by excessive in- generally consistent with the “Stanton–Pott hypothesis” flammatory responses to CNT fibers. that asbestos fibers with a diameter of ≤0.25 μmand MWCNTs and asbestos show similar genotoxicity char- length of ≥20 μm are highly carcinogenic [7, 8]. Muller acteristics even in cell culture experiments, and both are et al. reported no increase in carcinogenesis in Wistar rats known to induce polyploid cells (and multinucleated cells) following intraperitoneal administration of short carbon with a high frequency [12–15]. Chromosomal abnormal- nanotube (CNT) fibers whose length was less than that in- ities caused by polyploidization and aneuploidization alter dicated in the hypothesis (average length: ≤1 μm) [9]. Only the expression of a variety of genes involved in carcinogen- a limited number of in vivo studies have investigated the esis and thus are believed to be closely related to asbestos- genotoxicity of MWCNTs [10, 11]. Kato et al. [11] induced mesothelioma and bronchial cancer, as observed in animal studies [16, 17]. Jensen et al. conducted time- * Correspondence: m-yasui@nihs.go.jp lapse analysis using a microscope applicable for live-cell Division of Genetics and Mutagenesis, National Institute of Health Sciences, observation to determine the mechanisms underlying the 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan induction of abnormal binucleated and multinucleated Full list of author information is available at the end of the article © 2015 Yasui et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yasui et al. Genes and Environment (2015) 37:6 Page 2 of 6 cells by asbestos [18]. They observed that comparatively Live-cell imaging long crocidolite (15–50 μm) fibers were trapped in the We used an FV1000 laser fluorescence microscope contractile ring during anaphase in LLC-MK epithelial (Olympus Corp., Tokyo, Japan) equipped with a humid cells, which created a physical barrier to cytokinesis, even- chamber to capture images as the cells were cultured. tually causing formation of binucleated cells. On the other We also used a multi-Ar and He–Ne G laser and an ob- hand, many reports have demonstrated a causal role of jective lens with 60× magnification (1.20 Numerical MWCNTs in cell multinucleation and polyploidization; Aperture). For imaging, 5 × 10 MDA-435 cells were cul- however, only few have directly demonstrated the mechan- tured in 2 mL of DMEM containing 10 % fetal bovine ism underlying the occurrence of these aberrations [19]. serum (37 °C, 5 % CO , 100 % humidity) in a 35-mm In this study, we conducted time-lapse analysis with a glass base dish (IWAKI, ASAHI GLASS CO., Ltd., high-resolution confocal live-cell imaging system to elu- Tokyo, Japan). To minimize cytotoxicity of the laser, we cidate the mechanism involved in the MWCNT-induced conducted the experiments at a weak laser output such formation of binucleated cells using dichromatically vi- that ≥50 % cells divided after 24 h among the control sualized human cells in which chromosomes and centro- cells. The acquired images were edited using Volocity meres were stained with different fluorescent proteins. Software (PerkinElmer Inc., Massachusetts, USA), and We found that short CNT fibers (approximately 5 μm) the resulting moving images were analyzed. When migrated to either of the daughter cells immediately MWCNTs were added to the medium (final concentra- after chromosome segregation, whereas long fibers (ap- tion: 0, 12.7, 25.3, or 50.6 μg/mL), images of a visual proximately 20 μm) formed a bridge structure between field containing a large number of cells in metaphase the 2 daughter cells during anaphase and induced the were taken at 5-min intervals for a period of 48–72 h (in formation of binucleated cells by impeding cytokinesis. the z-axial direction, photographs were taken every This physical disruption of cytokinesis was very similar 2.0 μm). All cells in the visual field were counted for to the asbestos-induced disruption described above. each MWCNT concentration, and the incidences (%) of cells that completed cell division, cells that were unable Materials and methods to undergo cell division and subsequently died, and cells MWCNTs that became binucleated were calculated by dividing the The MWCNTs used in this study were MWCNT-7 (Lot number of such cells by the total cell count. We did not No.060125-01k) manufactured by Mitsui & Co., Ltd. take statistical analysis for the incidences of divided, (Ibaraki, Japan), which was same batch used in the study dead, and bi-nucleated cells, since the images of a visual reported by Takagi et al. [4]. According to the report, field containing a large number of cells in metaphase these MWCNT fibers were approximately 100 nm in were intentionally selected. Bi- and multi-nucleated cells diameter and contained 27.5 % of MWCNTs ≥5 μmin had more than two nuclei in a cell. The cell death was length. The MWCNTs were suspended in 100 % fetal defined as mitotic catastrophe during M-phase (from bovine serum (Gibco, Invitrogen, NY, USA) at a concen- prophase to telophase). Approximate length of MWCNT tration of 1 mg/mL and were autoclaved for 15 min at fiber was estimated from bar length given in each 121 °C. Thereafter, Tween 80 (Tokyo Chemical Industry images. Co., Ltd., Tokyo, Japan) was added to a final concentra- tion of 1.0 % in fetal bovine serum. The resulting mix- Results and discussion ture was subjected to ultrasonication using an ultrasonic Endocytosis of MWCNTs homogenizer (VP30s, TAITEC Co., Saitama, Japan). We conducted time-lapse imaging at the MWCNT con- centration of 50.6 μg/mL to determine how MWCNTs Cell culture undergo endocytosis. The results showed that some cells Dichromatically visualized MDA-435 human breast cancer actively ingested and incorporated CNT fibers within a cells, in which chromosomes and centromeres were stained few hours after addition of MWCNTs (Additional file 1, with a red fluorescent protein (mCherry–Histone H3 the right side in the movie) and other cells did not. Simi- fusion) and green fluorescent protein (EGFP–CENP-A fu- lar findings have been reported in a previous study in sion), respectively,werekindlyprovidedbyDr. KenjiSugi- which MWCNTs were easily incorporated within 24 h in moto (Osaka Prefecture University, Osaka, Japan) [20]. The experiments with human neonatal epidermal keratino- cells were cultured at 37 °C (5 % CO , 100 % humidity) in cytes [22]. CNT fibers that were incorporated into a cell Dulbecco’s Modified Eagle’s medium (DMEM) (Nacalai remained there for a long time without being ejected Tesque,Kyoto,Japan),supplemented with 10 % fetal bovine and migrated to either of the daughter cells after cell serum. MDA-435 cell line, isolated from ductal adenocar- division. In addition, we observed that CNT fibers were cinoma of female breast, is aneuploid with most chromo- frequently stuck to the cell surface. After 72 h, virtually some counts in the 55–60 range (modal number = 56) [21]. no abnormal cells (such as multinucleated cells) were Yasui et al. Genes and Environment (2015) 37:6 Page 3 of 6 Control CNT treatment CNT only Fig. 1 Comparison of untreated (control) and MWCNT (50.6 μg/mL) treated cells after 72 h. Many abnormal cells (such as multinucleated cells) were found in the MWCNT-treated cell group (the white dotted line in the image). Bar, 41 μm found in the untreated (control) group of cells (Fig. 1). 40 (38 %), 49 (27 %), and 34 (12 %) cells completed cell On the other hand, in the MWCNT-treated cell group, division and 4 (3.2 %), 11 (5.9 %), and 21 (7.6 %) cells most cells were entangled with CNT fibers, and many died at concentrations of 12.7, 25.3, and 50.6 μg/mL, multinucleated cells were observed (Fig. 1, the white respectively. Thus, the number of cells that completed dotted line). cell division decreased and the number of dead cells increased with an increase in MWCNT concentration, Cytotoxicity of MWCNTs and the incidence of binucleated indicating the concentration-dependent cytotoxicity of cells MWCNTs. However, endocytosis of MWCNT was dif- Time-lapse images of MDA-435 cells in the medium ferent depending on individual cells, as described above. containing MWCNTs (0, 12.7, 25.3, or 50.6 μg/mL) were Some cells did not undergo cell death when incor- taken up to 72 h. The experiment was conducted 3 porated a small number of CNT fibers. Actually, both times with each concentration, and 232, 112, 170, and incidences of divided cells between control and low 282 cells were imaged in the visual field, respectively concentration (12.7 μg/mL) were almost same (40 and (Table 1). Among the untreated cells, 93 (40 %) of the 38 %), as shown in Table 1. When ingested many CNT 232 cells completed cell division and only 4 (1.7 %) cells fibers even at low concentration, the cells gave rise to died during cell division. In the MWCNT-treated group, mitotic catastrophe. In addition, the number of Table 1 Observation of cell division involving MWCNTs using live-cell imaging MWCNTs (μg/mL) Experiment Total of recorded cells No. of divided cells No. of dead cells during mitosis No. of binucleated cells during mitosis 0 1 76 37 1 0 274 27 2 0 382 29 1 0 Total 232 93 (40 %) 4 (1.7 %) 0 12.7 1 33 16 0 0 232 13 1 0 347 11 3 0 Total 112 40 (38 %) 4 (3.2 %) 0 25.3 1 38 5 2 1 251 19 2 2 381 25 7 2 Total 170 49 (27 %) 11 (5.9 %) 5 (3.0 %) 50.6 1 86 12 10 1 2 101 11 5 0 395 11 6 0 Total 282 34 (12 %) 21 (7.6 %) 1 (0.4 %) Statistical analysis for the incidences of divided, dead, and binucleated cells was not done, as described in Materials and Methods Yasui et al. Genes and Environment (2015) 37:6 Page 4 of 6 binucleated cells was 0 (0 %), 5 (3 %), and 1 (0.4 %) at (the white arrow in Fig. 2a) and presumably exerted no the respective concentrations. A possible reason for the lethal damage during karyokinesis or cytokinesis. In lower incidence of binucleated cells at the highest dose contrast, cell division involving long CNT fibers (ap- of 50.6 μg/mL than at the lower dose of 25.3 μg/mL was proximately 20 μm) took almost 3 h (Fig. 2b). The long that the cells underwent cell division less frequently at CNT fibers formed a bridge between the 2 daughter the highest dose, as described above; furthermore, a very cells during anaphase (arrowheads in Fig. 2b), and large number of cells did not enter the mitotic phase remained in the contractile ring (2:30). Thereafter, and remained in interphase during imaging. karyokinesis was only slightly delayed and was com- pleted normally without micronuclei formation (2:50). Formation of binucleated cells through disturbance of However, approximately at the same time, cytokinesis cytokinesis was impeded by the CNT fiber bridge; consequently, the Time-lapse images of typical cell division involving constriction of the contractile ring was gradually MWCNTs are shown in Fig. 2. Normal cell division was abrogated (3:20). Thereafter, the borderline between the completed within 30 min of chromosome segregation 2 cells disappeared (4:40), resulting in the formation of during metaphase. Cell division involving short CNT fibers binucleated cells (5:40, the white dotted line). This dis- (approximately 5 μm) (Fig. 2a) occurred smoothly, similar ruption of cytokinesis was very similar to the process to that in the untreated group of cells, and was completed observed with crocidolite [18]. within 30 min. These short CNT fibers migrated to the MWCNTs used in this experiment contained approxi- daughter cells immediately after chromosome segregation mately 3500 ppm of iron and thus may have caused Fig. 2 Time-lapse analysis of cell division involving MWCNT fibers (25.3 μg/mL). a Short MWCNT fibers (approximately 5 μm) did not inhibit cell division. b Long MWCNT fibers (approximately 20 μm) inhibited cytokinesis and induced the formation of binucleated cells (the white dotted line in the image at 5 h 40 min). Time (h:min) is shown at the top. Bar, 41 μm Yasui et al. Genes and Environment (2015) 37:6 Page 5 of 6 oxidative DNA damage to the cell genome by the react- Pharmaceutical Sciences, Teikyo Heisei University, 4-21-2 Nakano, Nakano-ku, Tokyo 164-8530, Japan. ive oxygen species formed during the Fenton reaction [4, 23, 24]. Nonetheless, even with the analysis system Received: 5 November 2014 Accepted: 17 February 2015 used in this study capable of detecting extremely small micronuclei [25], we did not observe any abnormality (such as micronuclei formation or abnormal multipolar References 1. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, division involving multiple centromeres) attributable to et al. Carbon nanotubes introduced into the abdominal cavity of mice incubation with CNT fibers within the 72-h imaging show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. period. Asakura et al. used MWCNT-7 (iron content: 2008;3:423–8. 2. Toyokuni S. Genotoxicity and carcinogenicity risk of carbon nanotubes. 4400 ppm) obtained from the same manufacturer to Adv Drug Deliv Rev. 2013;65:2098–110. perform a chromosome abnormality test, an in vitro 3. Donaldson K, Poland CA, Murphy FA, MacFarlane M, Chernova T, Schinwald micronucleus test, and the Hprt gene mutation assay in A. Pulmonary toxicity of carbon nanotubes and asbestos - similarities and differences. Adv Drug Deliv Rev. 2013;65:2078–86. Chinese hamster lung (CHL/IU) cells. The test results 4. Takagi A, Hirose A, Nishimura T, Fukumori N, Ogata A, Ohashi N, et al. were all negative, but they observed an increase in the Induction of mesothelioma in p53+/− mouse by intraperitoneal application number of binucleated and polyploid cells with an of multi-wall carbon nanotube. J Toxicol Sci. 2008;33:105–16. 5. Takagi A, Hirose A, Futakuchi M, Tsuda H, Kanno J. Dose-dependent increase in MWCNT concentration [14]. These results mesothelioma induction by intraperitoneal administration of multi-wall are consistent with the mechanism of induction of binu- carbon nanotubes in p53 heterozygous mice. Cancer Sci. 2012;103:1440–4. cleated cells observed in this study and suggest that CNT 6. Sakamoto Y, Nakae D, Fukumori N, Tayama K, Maekawa A, Imai K, et al. Induction of mesothelioma by a single intrascrotal administration of fibers containing comparatively less iron result in greater multi-wall carbon nanotube in intact male Fischer 344 rats. J Toxicol Sci. physical disruption of cytokinesis than DNA damage by 2009;34:65–76. reactive oxygen species. In other words, MWCNTs may 7. Stanton MF, Layard M, Tegeris A, Miller E, May M, Morgan E, et al. Relation of particle dimension to carcinogenicity in amphibole asbestoses and other allow karyokinesis to proceed and may induce abnormal fibrous minerals. J Natl Cancer Inst. 1981;67:965–75. cells that are either binuclear or tetranuclear, considering 8. Pott F, Schlipkoter HW, Ziem U, Spurny K, Huth F. New results from implantation that MWCNTs inhibit cytokinesis but do not cause experiments with mineral fibres. In: Proceedings of a WHO/IARC Conference: Biological effects of man-made mineral fibres. Copenhagen: World Heath lethal damage to the nucleus or chromosomes to the Organization; 1984. p. 286–302. extent that prevents cell division (Fig. 1). 9. Muller J, Delos M, Panin N, Rabolli V, Huaux F, Lison D. Absence of In conclusion, we observed that comparatively long carcinogenic response to multiwall carbon nanotubes in a 2-year bioassay in the peritoneal cavity of the rat. Toxicol Sci. 2009;110:442–8. MWCNTs (approximately ≥20 μm) inhibited cytokinesis 10. Kim JS, Sung JH, Choi BG, Ryu HY, Song KS, Shin JH, et al. In vivo during cell division and induced the formation of genotoxicity evaluation of lung cells from Fischer 344 rats following 28 days binucleated cells, whereas short MWCNTs did not. of inhalation exposure to MWCNTs, plus 28 days and 90 days post-exposure. Inhal Toxicol. 2014;26:222–34. These results indicate that the mechanism of induction 11. Kato T, Totsuka Y, Ishino K, Matsumoto Y, Tada Y, Nakae D, et al. of binucleated cells by MWCNTs is very similar to that Genotoxicity of multi-walled carbon nanotubes in both in vitro and in vivo observed with crocidolite. assay systems. Nanotoxicology. 2013;7:452–61. 12. Jaurand MC, Bastie-Sigeac I, Renier A, Bignon J. Comparative toxicities of different forms of asbestos on rat pleural mesothelial cells. Environ Health Additional file Perspect. 1983;51:153–8. 13. Kenne K, Ljungquist S, Ringertz NR. Effects of asbestos fibers on cell division, cell survival, and formation of thioguanine-resistant mutants in Chinese Additional file 1: Time-lapse of endocytosis of cells exposed to hamster ovary cells. Environ Res. 1986;39:448–64. MWCNT (50.6 μg/mL). MWCNTs were easily incorporated within a few 14. Asakura M, Sasaki T, Sugiyama T, Takaya M, Koda S, Nagano K, et al. hours. CNT fibers that were incorporated into a cell remained there for a Genotoxicity and cytotoxicity of multi-wall carbon nanotubes in cultured long time without being ejected and migrated to either of the daughter Chinese hamster lung cells in comparison with chrysotile A fibers. J Occup cells after cell division. Bar, 41 μm. Health. 2010;52:155–66. 15. Muller J, Decordier I, Hoet PH, Lombaert N, Thomassen L, Huaux F, et al. Competing interests Clastogenic and aneugenic effects of multi-wall carbon nanotubes in The authors declare that they have no competing interest. epithelial cells. Carcinogenesis. 2008;29:427–33. 16. Oshimura M, Hesterberg TW, Tsutsui T, Barrett JC. Correlation of asbestos-induced Authors’ contributions cytogenetic effects with cell transformation of Syrian hamster embryo cells in MY and NK collected the data. TN prepared MWCNT samples. MY and MH culture. Cancer Res. 1984;44:5017–22. designed and critically discussed the study. All authors read and approved 17. Craighead JE, Akley NJ, Gould LB, Libbus BL. Characteristics of tumors and the final manuscript. tumor cells cultured from experimental asbestos-induced mesotheliomas in rats. Am J Pathol. 1987;129:448–62. Acknowledgments 18. Jensen CG, Jensen LC, Rieder CL, Cole RW, Ault JG. Long crocidolite We thank Dr. Kenji Sugimoto for the generous gift of dichromatically asbestos fibers cause polyploidy by sterically blocking cytokinesis. visualized MDA-435 cells. This study was supported by a Grant-in-Aid for Carcinogenesis. 1996;17:2013–21. Scientific Research from the Ministry of Health, Labour and Welfare 19. Sargent LM, Reynolds SH, Castranova V. Potential pulmonary effects of (H24-Food-General-011 and H24-kagaku-shitei-009). engineered carbon nanotubes: in vitro genotoxic effects. Nanotoxicology. 2010;4:396–408. Author details 20. Sugimoto K, Senda-Murata K, Oka S. Construction of three quadruple-fluorescent Division of Genetics and Mutagenesis, National Institute of Health Sciences, MDA435 cell lines that enable monitoring of the whole chromosome 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Faculty of segregation process in the living state. Mutat Res. 2008;657:56–62. Yasui et al. Genes and Environment (2015) 37:6 Page 6 of 6 21. Phillips KK, Welch DR, Miele ME, Lee JH, Wei LL, Weissman BE. Suppression of MDA-MB-435 breast carcinoma cell metastasis following the introduction of human chromosome 11. Cancer Res. 1996;56:1222–7. 22. Monteiro-Riviere NA, Nemanich RJ, Inman AO, Wang YY, Riviere JE. Multi-walled carbon nanotube interactions with human epidermal keratinocytes. Toxicol Lett. 2005;155:377–84. 23. Tabet L, Bussy C, Amara N, Setyan A, Grodet A, Rossi MJ, et al. Adverse effects of industrial multiwalled carbon nanotubes on human pulmonary cells. J Toxicol Environ Health A. 2009;72:60–73. 24. Kagan VE, Tyurina YY, Tyurin VA, Konduru NV, Potapovich AI, Osipov AN, et al. Direct and indirect effects of single walled carbon nanotubes on RAW 264.7 macrophages: role of iron. Toxicol Lett. 2006;165:88–100. 25. Yasui M, Koyama N, Koizumi T, Senda-Murata K, Takashima Y, Hayashi M, et al. Live cell imaging of micronucleus formation and development. Mutat Res. 2010;692:12–8. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

Journal

Genes and Environment – Springer Journals

Published: Jun 16, 2015

There are no references for this article.