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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 differentiation but increase cell death of ectodermal stem cells.Ventral views; Anterior is up. Bar equals 5 µm in A–F. Single neural stem cells were labelled with DiI (red) in embryos injected with 10% DMSO (DMSO; A, C, E) or with 1 mg/ml MWCNT in 10% DMSO/water (MWCNT/DMSO; B, D, F). The identification of the stem cell (1-2, 7-3, 3-1) is given on the left. (A–F) MWCNT does not interfere with neuronal differentiation. Labelled stem cells were allowed to divide and the progeny were examined for axonal extension (arrow), cell body position (arrowhead) and cell number. To examine the overall development of the CNS either all axons (anti-BP102; C, D) or all neuronal membranes, axons and cell body (anti-HRP; E, F) were immunostained. We do not detect any differences between stem cell progeny in non-injected embryos [34], DMSO or MWCNT/DMSO injected embryos. Note that the difference in branching pattern of 3-1 progeny (E, F) is caused by their different anterior-posterior location [34] and not MWCNT injections. (G) Frequency and kind of progeny obtained from labelled embryonic stem cells. Compared to non-injected embryos (grey), the injection of DMSO (yellow) results in an increase of cell death of neural stem cells. Injection of MWCNT/DMSO (black) affects the survival of epidermal as well as neural stem cells resulting in a significant (p<0.05) increase in cell death. The cell death increase observed in the case of neural stem cells is not higher than upon injection of DMSO only. Y-axis, frequency in % of epidermal (epidermis; column 1–3) or neural progeny (neural; column 4–6) or cell death (column 7–9) arisen from labelled stem cells; X-axis, total number of cells labelled (n). Numbers for non-injected embryo are taken from Bossing et al., 1996.
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pone-0088681-g004: MWCNTs do not interfere with differentiation but increase cell death of ectodermal stem cells.Ventral views; Anterior is up. Bar equals 5 µm in A–F. Single neural stem cells were labelled with DiI (red) in embryos injected with 10% DMSO (DMSO; A, C, E) or with 1 mg/ml MWCNT in 10% DMSO/water (MWCNT/DMSO; B, D, F). The identification of the stem cell (1-2, 7-3, 3-1) is given on the left. (A–F) MWCNT does not interfere with neuronal differentiation. Labelled stem cells were allowed to divide and the progeny were examined for axonal extension (arrow), cell body position (arrowhead) and cell number. To examine the overall development of the CNS either all axons (anti-BP102; C, D) or all neuronal membranes, axons and cell body (anti-HRP; E, F) were immunostained. We do not detect any differences between stem cell progeny in non-injected embryos [34], DMSO or MWCNT/DMSO injected embryos. Note that the difference in branching pattern of 3-1 progeny (E, F) is caused by their different anterior-posterior location [34] and not MWCNT injections. (G) Frequency and kind of progeny obtained from labelled embryonic stem cells. Compared to non-injected embryos (grey), the injection of DMSO (yellow) results in an increase of cell death of neural stem cells. Injection of MWCNT/DMSO (black) affects the survival of epidermal as well as neural stem cells resulting in a significant (p<0.05) increase in cell death. The cell death increase observed in the case of neural stem cells is not higher than upon injection of DMSO only. Y-axis, frequency in % of epidermal (epidermis; column 1–3) or neural progeny (neural; column 4–6) or cell death (column 7–9) arisen from labelled stem cells; X-axis, total number of cells labelled (n). Numbers for non-injected embryo are taken from Bossing et al., 1996.

Mentions: We injected MWCNTs and vehicle control (10%DMSO in water) into the syncytial blastoderm. 1.5 h after injection we labelled three to six cells at the injection site with the lipophilic dye DiI [21]. The dye is transferred from the stem cell to the progeny and labels the cell membrane of all progeny (Figure 4). We allowed the embryos to complete their development before we processed them for immunostaining and recorded the progeny. Consistent with our previous results (Figure 1), immunostaining against axonal or neuronal surface proteins did not show any gross morphological disturbances of the nervous sytem. This result is supported by the observed normal differentiation of epidermal and neural progeny derived from the labelled stem cells (Figure 4A–F). Ectodermal stem cells in non-injected embryos can give rise to 2–12 progeny with an average of 6.07 (+/−2.82, n = 202). In embryos injected with 10% DMSO progeny number varies between 2–11, average 6.04 (+/−2.56, n = 25) and injection of MWCNTs result in 2–12 daughter cells/ectodermal precursor, average 6.27 (+/−2.78, n = 48). The number of progeny derived from neural stem cells is stem cell specific [34] and we did not detect any differences between identical stem cells labelled in non-injected, 10% DMSO injected, and MWCNT injected embryos. We conclude that the presence of MWCNTs does not affect the number of progeny i.e. the division pattern of precursors cells.


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 differentiation but increase cell death of ectodermal stem cells.Ventral views; Anterior is up. Bar equals 5 µm in A–F. Single neural stem cells were labelled with DiI (red) in embryos injected with 10% DMSO (DMSO; A, C, E) or with 1 mg/ml MWCNT in 10% DMSO/water (MWCNT/DMSO; B, D, F). The identification of the stem cell (1-2, 7-3, 3-1) is given on the left. (A–F) MWCNT does not interfere with neuronal differentiation. Labelled stem cells were allowed to divide and the progeny were examined for axonal extension (arrow), cell body position (arrowhead) and cell number. To examine the overall development of the CNS either all axons (anti-BP102; C, D) or all neuronal membranes, axons and cell body (anti-HRP; E, F) were immunostained. We do not detect any differences between stem cell progeny in non-injected embryos [34], DMSO or MWCNT/DMSO injected embryos. Note that the difference in branching pattern of 3-1 progeny (E, F) is caused by their different anterior-posterior location [34] and not MWCNT injections. (G) Frequency and kind of progeny obtained from labelled embryonic stem cells. Compared to non-injected embryos (grey), the injection of DMSO (yellow) results in an increase of cell death of neural stem cells. Injection of MWCNT/DMSO (black) affects the survival of epidermal as well as neural stem cells resulting in a significant (p<0.05) increase in cell death. The cell death increase observed in the case of neural stem cells is not higher than upon injection of DMSO only. Y-axis, frequency in % of epidermal (epidermis; column 1–3) or neural progeny (neural; column 4–6) or cell death (column 7–9) arisen from labelled stem cells; X-axis, total number of cells labelled (n). Numbers for non-injected embryo are taken from Bossing et al., 1996.
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pone-0088681-g004: MWCNTs do not interfere with differentiation but increase cell death of ectodermal stem cells.Ventral views; Anterior is up. Bar equals 5 µm in A–F. Single neural stem cells were labelled with DiI (red) in embryos injected with 10% DMSO (DMSO; A, C, E) or with 1 mg/ml MWCNT in 10% DMSO/water (MWCNT/DMSO; B, D, F). The identification of the stem cell (1-2, 7-3, 3-1) is given on the left. (A–F) MWCNT does not interfere with neuronal differentiation. Labelled stem cells were allowed to divide and the progeny were examined for axonal extension (arrow), cell body position (arrowhead) and cell number. To examine the overall development of the CNS either all axons (anti-BP102; C, D) or all neuronal membranes, axons and cell body (anti-HRP; E, F) were immunostained. We do not detect any differences between stem cell progeny in non-injected embryos [34], DMSO or MWCNT/DMSO injected embryos. Note that the difference in branching pattern of 3-1 progeny (E, F) is caused by their different anterior-posterior location [34] and not MWCNT injections. (G) Frequency and kind of progeny obtained from labelled embryonic stem cells. Compared to non-injected embryos (grey), the injection of DMSO (yellow) results in an increase of cell death of neural stem cells. Injection of MWCNT/DMSO (black) affects the survival of epidermal as well as neural stem cells resulting in a significant (p<0.05) increase in cell death. The cell death increase observed in the case of neural stem cells is not higher than upon injection of DMSO only. Y-axis, frequency in % of epidermal (epidermis; column 1–3) or neural progeny (neural; column 4–6) or cell death (column 7–9) arisen from labelled stem cells; X-axis, total number of cells labelled (n). Numbers for non-injected embryo are taken from Bossing et al., 1996.
Mentions: We injected MWCNTs and vehicle control (10%DMSO in water) into the syncytial blastoderm. 1.5 h after injection we labelled three to six cells at the injection site with the lipophilic dye DiI [21]. The dye is transferred from the stem cell to the progeny and labels the cell membrane of all progeny (Figure 4). We allowed the embryos to complete their development before we processed them for immunostaining and recorded the progeny. Consistent with our previous results (Figure 1), immunostaining against axonal or neuronal surface proteins did not show any gross morphological disturbances of the nervous sytem. This result is supported by the observed normal differentiation of epidermal and neural progeny derived from the labelled stem cells (Figure 4A–F). Ectodermal stem cells in non-injected embryos can give rise to 2–12 progeny with an average of 6.07 (+/−2.82, n = 202). In embryos injected with 10% DMSO progeny number varies between 2–11, average 6.04 (+/−2.56, n = 25) and injection of MWCNTs result in 2–12 daughter cells/ectodermal precursor, average 6.27 (+/−2.78, n = 48). The number of progeny derived from neural stem cells is stem cell specific [34] and we did not detect any differences between identical stem cells labelled in non-injected, 10% DMSO injected, and MWCNT injected embryos. We conclude that the presence of MWCNTs does not affect the number of progeny i.e. the division pattern of precursors cells.

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