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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 gross embryonic development and viability.Ventral views; Anterior is up. Bar, 10 µm. (A) Using a microcapillary, we injected DiI labelled MWCNTs (red) into the syncytial blastoderm of Drosophila embryos. At this stage of development, all nuclei (green) share the same cytoplasm permitting an unhindered diffusion (arrows) of the MWCNTs to nuclei adjacent to the injection site. In the next hour of embryogenesis, invaginating cell membranes will partition the nuclei into single cells incorporating the MWCNTs. (B, C) Live snapshots of embryos injected with 1 mg/ml DiI in DMSO (red, B, DMSO/DiI) or 1 mg/ml DiI labelled MWCNTs in DMSO (red, C, MWCNT-DiI) immediately after injection into an embryo with microtubules labelled by GFP (green, GFP-jupiter). MWCNTs remain in the cytoplasm, the darker area surrounding the microtubules. Note that DiI not bound to MWCNT shows a diffuse membrane stain, whereas DiI bound to MWCNTs can be detected as small puncta (arrow). (D, E) Live confocal section of the same embryo 8 h (D) and 15 h (E) after injection. Cell outlines are labelled by GFP labelled microtubules (green, Jupiter-GFP). The majority of MWCNT (red) has been incorporated into the newly formed epidermis cells (arrows). (F, G) Live confocal section of the developing CNS of the same embryo 8 h (F) and 15 h (G) after injection. Cell outline and axons are labelled by GFP labelled microtubules (green, Jupiter-GFP). MWCNTs do not interfere with the initial outgrowth of axons (F, arrows) and are incorporated into the axonal scaffold without visible damage (G, arrows). (H) MWCNT injections do not interfere with embryonic viability. If embryos develop into healthy larvae, the larvae will hatch out of the egg shell. Hatching rate of embryos injected with water (blue, injection control), 10% DMSO (yellow, vehicle control), 1 mg/ml MWCNT in 10% DMSO/water (black, MWCNT/DMSO, see also Experimental Section) and 1 mg/ml MWCNT in 10% DMSO labelled with DiI (red, MWCNT/DiI/DMSO). Pairwise comparison with water injected embryos (t-Test), *, significant (p<0.05); error bars, StDev;. Y-axis, Hatching rate in % of injected embryos; X-axis, Number of injected embryos (n).
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pone-0088681-g001: MWCNTs do not interfere with gross embryonic development and viability.Ventral views; Anterior is up. Bar, 10 µm. (A) Using a microcapillary, we injected DiI labelled MWCNTs (red) into the syncytial blastoderm of Drosophila embryos. At this stage of development, all nuclei (green) share the same cytoplasm permitting an unhindered diffusion (arrows) of the MWCNTs to nuclei adjacent to the injection site. In the next hour of embryogenesis, invaginating cell membranes will partition the nuclei into single cells incorporating the MWCNTs. (B, C) Live snapshots of embryos injected with 1 mg/ml DiI in DMSO (red, B, DMSO/DiI) or 1 mg/ml DiI labelled MWCNTs in DMSO (red, C, MWCNT-DiI) immediately after injection into an embryo with microtubules labelled by GFP (green, GFP-jupiter). MWCNTs remain in the cytoplasm, the darker area surrounding the microtubules. Note that DiI not bound to MWCNT shows a diffuse membrane stain, whereas DiI bound to MWCNTs can be detected as small puncta (arrow). (D, E) Live confocal section of the same embryo 8 h (D) and 15 h (E) after injection. Cell outlines are labelled by GFP labelled microtubules (green, Jupiter-GFP). The majority of MWCNT (red) has been incorporated into the newly formed epidermis cells (arrows). (F, G) Live confocal section of the developing CNS of the same embryo 8 h (F) and 15 h (G) after injection. Cell outline and axons are labelled by GFP labelled microtubules (green, Jupiter-GFP). MWCNTs do not interfere with the initial outgrowth of axons (F, arrows) and are incorporated into the axonal scaffold without visible damage (G, arrows). (H) MWCNT injections do not interfere with embryonic viability. If embryos develop into healthy larvae, the larvae will hatch out of the egg shell. Hatching rate of embryos injected with water (blue, injection control), 10% DMSO (yellow, vehicle control), 1 mg/ml MWCNT in 10% DMSO/water (black, MWCNT/DMSO, see also Experimental Section) and 1 mg/ml MWCNT in 10% DMSO labelled with DiI (red, MWCNT/DiI/DMSO). Pairwise comparison with water injected embryos (t-Test), *, significant (p<0.05); error bars, StDev;. Y-axis, Hatching rate in % of injected embryos; X-axis, Number of injected embryos (n).

Mentions: Drosophila embryogenesis starts with the syncytial blastoderm when embryos only consist of nuclei, which undergo 13 near synchronous divisions. Before the last 4 divisions, nuclei align along the embryo surface. After the last nuclear division, an actin-based movement results in the ingression of cell membranes from the egg membrane, and each nuclei and its surrounding cytoplasm is partitioned into a newly formed cell [24]. We injected MWCNTs and vehicle controls at the time when nuclei reach the embryonic periphery (Figure 1A). MWCNTs were marked with the red fluorescent lipophilic dye DiI (Invitrogen) as this dye has been shown to bind non-covalently to carbon nanotubes [25] and to be harmless for Drosophila embryogenesis [21]. MWCNTs were labelled at 1 mg/ml DiI in 100% DMSO. DiI in 100% DMSO forms a homogenous solution, which does not precipitate when spun at room temperature at 6000 rpm. However, addition of MWCNT results in the formation of a reddish-black precipitate. Microscopic inspection of this precipitate under epifluorescence visualises small fluorescent puncta with the characteristic shape of MWCNTs. This observation concurs with previous reports showing binding of DiI to CNTs with high affinity [25]. MWCNT/DiI was injected as a colloidal suspension in 10% DMSO/water (see Experimental Section). As control we injected 100 µg/ml DiI in 50% DMSO to visualise the spread and accumulation of dye not bound to MWCNTs (Figure 1B). In contrast to 1 mg/ml DiI in DMSO, which diffused easily throughout the embryo labelling internal membranes (Figure 1B), injected MWCNTs formed small puncta, which only dispersed throughout four segments in the ventral half of the embryo (Figure 1C), equalling about 6% of total embryonic volume. The emission of unbound dye is no longer detectable after three nuclear divisions (data not shown) but the dye labelled MWCNT puncta can be followed throughout embryogenesis (Figure 1D–G) indicating that the dye remains bound to MWCNTs and does not diffuse away.


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 gross embryonic development and viability.Ventral views; Anterior is up. Bar, 10 µm. (A) Using a microcapillary, we injected DiI labelled MWCNTs (red) into the syncytial blastoderm of Drosophila embryos. At this stage of development, all nuclei (green) share the same cytoplasm permitting an unhindered diffusion (arrows) of the MWCNTs to nuclei adjacent to the injection site. In the next hour of embryogenesis, invaginating cell membranes will partition the nuclei into single cells incorporating the MWCNTs. (B, C) Live snapshots of embryos injected with 1 mg/ml DiI in DMSO (red, B, DMSO/DiI) or 1 mg/ml DiI labelled MWCNTs in DMSO (red, C, MWCNT-DiI) immediately after injection into an embryo with microtubules labelled by GFP (green, GFP-jupiter). MWCNTs remain in the cytoplasm, the darker area surrounding the microtubules. Note that DiI not bound to MWCNT shows a diffuse membrane stain, whereas DiI bound to MWCNTs can be detected as small puncta (arrow). (D, E) Live confocal section of the same embryo 8 h (D) and 15 h (E) after injection. Cell outlines are labelled by GFP labelled microtubules (green, Jupiter-GFP). The majority of MWCNT (red) has been incorporated into the newly formed epidermis cells (arrows). (F, G) Live confocal section of the developing CNS of the same embryo 8 h (F) and 15 h (G) after injection. Cell outline and axons are labelled by GFP labelled microtubules (green, Jupiter-GFP). MWCNTs do not interfere with the initial outgrowth of axons (F, arrows) and are incorporated into the axonal scaffold without visible damage (G, arrows). (H) MWCNT injections do not interfere with embryonic viability. If embryos develop into healthy larvae, the larvae will hatch out of the egg shell. Hatching rate of embryos injected with water (blue, injection control), 10% DMSO (yellow, vehicle control), 1 mg/ml MWCNT in 10% DMSO/water (black, MWCNT/DMSO, see also Experimental Section) and 1 mg/ml MWCNT in 10% DMSO labelled with DiI (red, MWCNT/DiI/DMSO). Pairwise comparison with water injected embryos (t-Test), *, significant (p<0.05); error bars, StDev;. Y-axis, Hatching rate in % of injected embryos; X-axis, Number of injected embryos (n).
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pone-0088681-g001: MWCNTs do not interfere with gross embryonic development and viability.Ventral views; Anterior is up. Bar, 10 µm. (A) Using a microcapillary, we injected DiI labelled MWCNTs (red) into the syncytial blastoderm of Drosophila embryos. At this stage of development, all nuclei (green) share the same cytoplasm permitting an unhindered diffusion (arrows) of the MWCNTs to nuclei adjacent to the injection site. In the next hour of embryogenesis, invaginating cell membranes will partition the nuclei into single cells incorporating the MWCNTs. (B, C) Live snapshots of embryos injected with 1 mg/ml DiI in DMSO (red, B, DMSO/DiI) or 1 mg/ml DiI labelled MWCNTs in DMSO (red, C, MWCNT-DiI) immediately after injection into an embryo with microtubules labelled by GFP (green, GFP-jupiter). MWCNTs remain in the cytoplasm, the darker area surrounding the microtubules. Note that DiI not bound to MWCNT shows a diffuse membrane stain, whereas DiI bound to MWCNTs can be detected as small puncta (arrow). (D, E) Live confocal section of the same embryo 8 h (D) and 15 h (E) after injection. Cell outlines are labelled by GFP labelled microtubules (green, Jupiter-GFP). The majority of MWCNT (red) has been incorporated into the newly formed epidermis cells (arrows). (F, G) Live confocal section of the developing CNS of the same embryo 8 h (F) and 15 h (G) after injection. Cell outline and axons are labelled by GFP labelled microtubules (green, Jupiter-GFP). MWCNTs do not interfere with the initial outgrowth of axons (F, arrows) and are incorporated into the axonal scaffold without visible damage (G, arrows). (H) MWCNT injections do not interfere with embryonic viability. If embryos develop into healthy larvae, the larvae will hatch out of the egg shell. Hatching rate of embryos injected with water (blue, injection control), 10% DMSO (yellow, vehicle control), 1 mg/ml MWCNT in 10% DMSO/water (black, MWCNT/DMSO, see also Experimental Section) and 1 mg/ml MWCNT in 10% DMSO labelled with DiI (red, MWCNT/DiI/DMSO). Pairwise comparison with water injected embryos (t-Test), *, significant (p<0.05); error bars, StDev;. Y-axis, Hatching rate in % of injected embryos; X-axis, Number of injected embryos (n).
Mentions: Drosophila embryogenesis starts with the syncytial blastoderm when embryos only consist of nuclei, which undergo 13 near synchronous divisions. Before the last 4 divisions, nuclei align along the embryo surface. After the last nuclear division, an actin-based movement results in the ingression of cell membranes from the egg membrane, and each nuclei and its surrounding cytoplasm is partitioned into a newly formed cell [24]. We injected MWCNTs and vehicle controls at the time when nuclei reach the embryonic periphery (Figure 1A). MWCNTs were marked with the red fluorescent lipophilic dye DiI (Invitrogen) as this dye has been shown to bind non-covalently to carbon nanotubes [25] and to be harmless for Drosophila embryogenesis [21]. MWCNTs were labelled at 1 mg/ml DiI in 100% DMSO. DiI in 100% DMSO forms a homogenous solution, which does not precipitate when spun at room temperature at 6000 rpm. However, addition of MWCNT results in the formation of a reddish-black precipitate. Microscopic inspection of this precipitate under epifluorescence visualises small fluorescent puncta with the characteristic shape of MWCNTs. This observation concurs with previous reports showing binding of DiI to CNTs with high affinity [25]. MWCNT/DiI was injected as a colloidal suspension in 10% DMSO/water (see Experimental Section). As control we injected 100 µg/ml DiI in 50% DMSO to visualise the spread and accumulation of dye not bound to MWCNTs (Figure 1B). In contrast to 1 mg/ml DiI in DMSO, which diffused easily throughout the embryo labelling internal membranes (Figure 1B), injected MWCNTs formed small puncta, which only dispersed throughout four segments in the ventral half of the embryo (Figure 1C), equalling about 6% of total embryonic volume. The emission of unbound dye is no longer detectable after three nuclear divisions (data not shown) but the dye labelled MWCNT puncta can be followed throughout embryogenesis (Figure 1D–G) indicating that the dye remains bound to MWCNTs and does not diffuse away.

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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Related in: MedlinePlus