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Drosophila embryos as model to assess cellular and developmental toxicity of multi-walled carbon nanotubes (MWCNT) in living organisms.

Liu B, Campo EM, Bossing T - PLoS ONE (2014)

Bottom Line: Analysis of developing ectodermal and neural stem cells in MWCNTs injected embryos revealed normal division patterns and differentiation capacity.However, an increase in cell death of ectodermal but not of neural stem cells was observed, indicating stem cell-specific vulnerability to MWCNT exposure.The ease of CNT embryo injections, the possibility of detailed morphological and genomic analysis and the low costs make Drosophila embryos a system of choice to assess potential developmental and cellular effects of CNTs and test their use in future CNT based new therapies including drug delivery.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Bangor, Bangor, United Kingdom.

ABSTRACT
Different toxicity tests for carbon nanotubes (CNT) have been developed to assess their impact on human health and on aquatic and terrestrial animal and plant life. We present a new model, the fruit fly Drosophila embryo offering the opportunity for rapid, inexpensive and detailed analysis of CNTs toxicity during embryonic development. We show that injected DiI labelled multi-walled carbon nanotubes (MWCNTs) become incorporated into cells in early Drosophila embryos, allowing the study of the consequences of cellular uptake of CNTs on cell communication, tissue and organ formation in living embryos. Fluorescently labelled subcellular structures showed that MWCNTs remained cytoplasmic and were excluded from the nucleus. Analysis of developing ectodermal and neural stem cells in MWCNTs injected embryos revealed normal division patterns and differentiation capacity. However, an increase in cell death of ectodermal but not of neural stem cells was observed, indicating stem cell-specific vulnerability to MWCNT exposure. The ease of CNT embryo injections, the possibility of detailed morphological and genomic analysis and the low costs make Drosophila embryos a system of choice to assess potential developmental and cellular effects of CNTs and test their use in future CNT based new therapies including drug delivery.

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MWCNTs do not interfere with cell division.Ventral views; Anterior is up. Bar equals 10 µm in A, B. (A) Single live confocal section through the syncytial blastoderm during a division wave. Microtubules are labelled with GFP (green, Jupiter-GFP). All nuclei are in the same stage (metaphase) of the cell cycle even if MWCNTs (red, arrows) are present. (B) Single live confocal section through an embryo with YFP labelled histones marking chromosomes (green). Regardless of the presence or absence of MWCNTs (red, arrows), all nuclei are in the same stage of division (anaphase). (C) The rate of nuclear divisions is not affected by the presence of MWCNTs. Division times for three nuclei per embryo half were recorded and division times between non-injected (non-hatched) and injected (hatched) halves compared. For non-injected embryos (grey) division times of three randomly chosen nuclei for each half were recorded. We detect no differences in the division times between non-injected and injected halves nor between embryos injected with 10% DMSO (yellow, solvent control), 1 mg/ml MWCNT in 10% DMSO/water (black, MWCNT/DMSO) and 1 mg/ml MWCNT in 10% DMSO labelled with DiI (red, MWCNT/DiI/DMSO). Y-axis, division time in minutes; X-axis, Number of nuclei analysed (n).
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pone-0088681-g002: MWCNTs do not interfere with cell division.Ventral views; Anterior is up. Bar equals 10 µm in A, B. (A) Single live confocal section through the syncytial blastoderm during a division wave. Microtubules are labelled with GFP (green, Jupiter-GFP). All nuclei are in the same stage (metaphase) of the cell cycle even if MWCNTs (red, arrows) are present. (B) Single live confocal section through an embryo with YFP labelled histones marking chromosomes (green). Regardless of the presence or absence of MWCNTs (red, arrows), all nuclei are in the same stage of division (anaphase). (C) The rate of nuclear divisions is not affected by the presence of MWCNTs. Division times for three nuclei per embryo half were recorded and division times between non-injected (non-hatched) and injected (hatched) halves compared. For non-injected embryos (grey) division times of three randomly chosen nuclei for each half were recorded. We detect no differences in the division times between non-injected and injected halves nor between embryos injected with 10% DMSO (yellow, solvent control), 1 mg/ml MWCNT in 10% DMSO/water (black, MWCNT/DMSO) and 1 mg/ml MWCNT in 10% DMSO labelled with DiI (red, MWCNT/DiI/DMSO). Y-axis, division time in minutes; X-axis, Number of nuclei analysed (n).

Mentions: The synchronised nuclear divisions in the syncytial blastoderm of Drosophila embryos offers the unique opportunity to check if the presence of MWCNTs interferes with DNA replication, and thereby slowing down or speeding up divisions. In addition, failure in DNA replication or chromosomal separation can easily be detected because these nuclei fall out of the embryonic surface into the yolk [28]. We injected MWCNTs and control vehicles into the syncytial blastoderm and recorded the last four synchronous nuclear divisions along the surface of the embryo (Movie S5, S6, S7). Nuclei surrounded by MWCNTs divided at the same time as adjacent MWCNT-free nuclei (Figure 2A, B). We did not detect any difference in the division cycles between control and MWCNT injected embryos or between injected and non-injected embryo halves (Figure 2C). We also did not observe any increase in nuclear fallout between embryos injected with MWCNTs or controls (data not shown). We conclude that MWCNTs do not interfere with DNA replication or chromosome separation. Interestingly, we never detected a nuclear incorporation of MWCNTs although the breakdown of the nuclear membrane during nuclear divisions would permit MWCNT entry. This argues for an yet to be described mechanism, which actively prevents the nuclear entry of MWCNTs.


Drosophila embryos as model to assess cellular and developmental toxicity of multi-walled carbon nanotubes (MWCNT) in living organisms.

Liu B, Campo EM, Bossing T - PLoS ONE (2014)

MWCNTs do not interfere with cell division.Ventral views; Anterior is up. Bar equals 10 µm in A, B. (A) Single live confocal section through the syncytial blastoderm during a division wave. Microtubules are labelled with GFP (green, Jupiter-GFP). All nuclei are in the same stage (metaphase) of the cell cycle even if MWCNTs (red, arrows) are present. (B) Single live confocal section through an embryo with YFP labelled histones marking chromosomes (green). Regardless of the presence or absence of MWCNTs (red, arrows), all nuclei are in the same stage of division (anaphase). (C) The rate of nuclear divisions is not affected by the presence of MWCNTs. Division times for three nuclei per embryo half were recorded and division times between non-injected (non-hatched) and injected (hatched) halves compared. For non-injected embryos (grey) division times of three randomly chosen nuclei for each half were recorded. We detect no differences in the division times between non-injected and injected halves nor between embryos injected with 10% DMSO (yellow, solvent control), 1 mg/ml MWCNT in 10% DMSO/water (black, MWCNT/DMSO) and 1 mg/ml MWCNT in 10% DMSO labelled with DiI (red, MWCNT/DiI/DMSO). Y-axis, division time in minutes; X-axis, Number of nuclei analysed (n).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928276&req=5

pone-0088681-g002: MWCNTs do not interfere with cell division.Ventral views; Anterior is up. Bar equals 10 µm in A, B. (A) Single live confocal section through the syncytial blastoderm during a division wave. Microtubules are labelled with GFP (green, Jupiter-GFP). All nuclei are in the same stage (metaphase) of the cell cycle even if MWCNTs (red, arrows) are present. (B) Single live confocal section through an embryo with YFP labelled histones marking chromosomes (green). Regardless of the presence or absence of MWCNTs (red, arrows), all nuclei are in the same stage of division (anaphase). (C) The rate of nuclear divisions is not affected by the presence of MWCNTs. Division times for three nuclei per embryo half were recorded and division times between non-injected (non-hatched) and injected (hatched) halves compared. For non-injected embryos (grey) division times of three randomly chosen nuclei for each half were recorded. We detect no differences in the division times between non-injected and injected halves nor between embryos injected with 10% DMSO (yellow, solvent control), 1 mg/ml MWCNT in 10% DMSO/water (black, MWCNT/DMSO) and 1 mg/ml MWCNT in 10% DMSO labelled with DiI (red, MWCNT/DiI/DMSO). Y-axis, division time in minutes; X-axis, Number of nuclei analysed (n).
Mentions: The synchronised nuclear divisions in the syncytial blastoderm of Drosophila embryos offers the unique opportunity to check if the presence of MWCNTs interferes with DNA replication, and thereby slowing down or speeding up divisions. In addition, failure in DNA replication or chromosomal separation can easily be detected because these nuclei fall out of the embryonic surface into the yolk [28]. We injected MWCNTs and control vehicles into the syncytial blastoderm and recorded the last four synchronous nuclear divisions along the surface of the embryo (Movie S5, S6, S7). Nuclei surrounded by MWCNTs divided at the same time as adjacent MWCNT-free nuclei (Figure 2A, B). We did not detect any difference in the division cycles between control and MWCNT injected embryos or between injected and non-injected embryo halves (Figure 2C). We also did not observe any increase in nuclear fallout between embryos injected with MWCNTs or controls (data not shown). We conclude that MWCNTs do not interfere with DNA replication or chromosome separation. Interestingly, we never detected a nuclear incorporation of MWCNTs although the breakdown of the nuclear membrane during nuclear divisions would permit MWCNT entry. This argues for an yet to be described mechanism, which actively prevents the nuclear entry of MWCNTs.

Bottom Line: Analysis of developing ectodermal and neural stem cells in MWCNTs injected embryos revealed normal division patterns and differentiation capacity.However, an increase in cell death of ectodermal but not of neural stem cells was observed, indicating stem cell-specific vulnerability to MWCNT exposure.The ease of CNT embryo injections, the possibility of detailed morphological and genomic analysis and the low costs make Drosophila embryos a system of choice to assess potential developmental and cellular effects of CNTs and test their use in future CNT based new therapies including drug delivery.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Bangor, Bangor, United Kingdom.

ABSTRACT
Different toxicity tests for carbon nanotubes (CNT) have been developed to assess their impact on human health and on aquatic and terrestrial animal and plant life. We present a new model, the fruit fly Drosophila embryo offering the opportunity for rapid, inexpensive and detailed analysis of CNTs toxicity during embryonic development. We show that injected DiI labelled multi-walled carbon nanotubes (MWCNTs) become incorporated into cells in early Drosophila embryos, allowing the study of the consequences of cellular uptake of CNTs on cell communication, tissue and organ formation in living embryos. Fluorescently labelled subcellular structures showed that MWCNTs remained cytoplasmic and were excluded from the nucleus. Analysis of developing ectodermal and neural stem cells in MWCNTs injected embryos revealed normal division patterns and differentiation capacity. However, an increase in cell death of ectodermal but not of neural stem cells was observed, indicating stem cell-specific vulnerability to MWCNT exposure. The ease of CNT embryo injections, the possibility of detailed morphological and genomic analysis and the low costs make Drosophila embryos a system of choice to assess potential developmental and cellular effects of CNTs and test their use in future CNT based new therapies including drug delivery.

Show MeSH
Related in: MedlinePlus