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Size effects of single-walled carbon nanotubes on in vivo and in vitro pulmonary toxicity.

Fujita K, Fukuda M, Endoh S, Maru J, Kato H, Nakamura A, Shinohara N, Uchino K, Honda K - Inhal Toxicol (2015)

Bottom Line: Comprehensive gene expression analysis confirmed that CNT-1-induced genes were strongly associated with inflammatory responses, cell proliferation, and immune system processes at 7 or 30 d post-instillation.Numerous genes were significantly upregulated or downregulated by CNT-2 at 1 d post-instillation.CNT-2 treatment induced cell growth inhibition, reactive oxygen species production, MIP-1α expression, and several genes involved in response to stimulus, whereas CNT-1 treatment did not exert a significant impact in these regards.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Science for Safety and Sustainability (RISS), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba, Ibaraki , Japan .

ABSTRACT
To elucidate the effect of size on the pulmonary toxicity of single-wall carbon nanotubes (SWCNTs), we prepared two types of dispersed SWCNTs, namely relatively thin bundles with short linear shapes (CNT-1) and thick bundles with long linear shapes (CNT-2), and conducted rat intratracheal instillation tests and in vitro cell-based assays using NR8383 rat alveolar macrophages. Total protein levels, MIP-1α expression, cell counts in BALF, and histopathological examinations revealed that CNT-1 caused pulmonary inflammation and slower recovery and that CNT-2 elicited acute lung inflammation shortly after their instillation. Comprehensive gene expression analysis confirmed that CNT-1-induced genes were strongly associated with inflammatory responses, cell proliferation, and immune system processes at 7 or 30 d post-instillation. Numerous genes were significantly upregulated or downregulated by CNT-2 at 1 d post-instillation. In vitro assays demonstrated that CNT-1 and CNT-2 SWCNTs were phagocytized by NR8383 cells. CNT-2 treatment induced cell growth inhibition, reactive oxygen species production, MIP-1α expression, and several genes involved in response to stimulus, whereas CNT-1 treatment did not exert a significant impact in these regards. These results suggest that SWCNTs formed as relatively thin bundles with short linear shapes elicited delayed pulmonary inflammation with slower recovery. In contrast, SWCNTs with a relatively thick bundle and long linear shapes sensitively induced cellular responses in alveolar macrophages and elicited acute lung inflammation shortly after inhalation. We conclude that the pulmonary toxicity of SWCNTs is closely associated with the size of the bundles. These physical parameters are useful for risk assessment and management of SWCNTs.

No MeSH data available.


Related in: MedlinePlus

Characterization of SWCNTs dispersed in working solutions. Characterization of SWCNTs in working solutions for in vivo and in vitro tests (A). TEM images of SWCNTs in CNT-1 (B and C) and CNT-2 (D and E) in working solutions for in vivo (high dose) studies. High-magnification micrographs (C and E). Distribution of SWCNT length in working solutions for in vivo (high dose) evaluation by digital TEM images (F and G).
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Figure 0001: Characterization of SWCNTs dispersed in working solutions. Characterization of SWCNTs in working solutions for in vivo and in vitro tests (A). TEM images of SWCNTs in CNT-1 (B and C) and CNT-2 (D and E) in working solutions for in vivo (high dose) studies. High-magnification micrographs (C and E). Distribution of SWCNT length in working solutions for in vivo (high dose) evaluation by digital TEM images (F and G).

Mentions: We prepared two types of dispersions of SWCNTs with different sizes to elucidate the effect of size on in vivo and in vitro toxicities. The characterization of SWCNTs in working solutions for in vivo and in vitro tests is shown in Figure 1. Analysis with an absorption spectrometer demonstrated nearly equivalent SWCNT concentrations in the adjusted working solutions (approximately 0.1 mg/mL and 1.0 mg/mL for in vivo tests; approximately 0.1 mg/mL for in vitro cell-based assays). DLS and zeta-potential studies revealed the average diameters of the dispersed SWCNT particles and the dispersibility/aggregability characteristics of CNT-1 and CNT-2. These analyses confirmed that SWCNT particles were stable and dispersed in the working solutions. DLS results confirmed that no significant changes occurred in the light scattering intensity-averaged diameter of SWCNTs in cell culture medium for 3 d after preparation (data not shown); thus, working solutions were used within 3 d after preparation for in vitro cell-based assays. Furthermore, no significant difference was found in the average diameter of SWCNTs in both stock suspensions stored at 4 °C for 8 weeks after preparation (data not shown). Working solutions were used within 7 d after preparation for in vivo tests. The negative zeta potential of all adjusted samples revealed that SWCNT particles were well dispersed in working solutions. Raman spectrophotometric analysis revealed that the G/D ratio for bulk SWCNTs was reduced slightly, from 5.9 to 4.1 or 2.4 for CNT-1 or CNT-2, respectively. ICP-MS results revealed that the metal content (e.g. Fe, Y, and Ni) of CNT-1 and CNT-2 was at an undetectable level. Previously, we demonstrated that the adsorption of proteins by ultrafine metal oxide particles influences their cytotoxicity to cultured cells (Horie et al., 2009). There was no significant reduction of salt concentrations (e.g. Na, P, and Ca) in CNT-1 and CNT-2 in culture medium (data not shown). TEM images of representative SWCNT dispersions showed differences in their lengths in working solutions, designated here as CNT-1 and CNT-2. CNT-1 was observed to contain relatively thick SWCNT structures with a short linear shape (Figure 1A, B, and F). The nanotubes in the CNT-2 working solution assembled into relatively thick bundles of SWCNTs with a long linear shape (Figure 1D, E, and G). Noticeably aggregated forms of SWCNTs in each working solution were not identified. These studies revealed that CNT-1 and CNT-2 were stably dispersed and assembled into relatively thick bundles with a long linear shape (CNT-1) or into relatively thin bundles with a short linear shape (CNT-2) in working solutions. The preparation procedure described in this study may thus be valuable for use in both in vivo and in vivo studies.Figure 1.


Size effects of single-walled carbon nanotubes on in vivo and in vitro pulmonary toxicity.

Fujita K, Fukuda M, Endoh S, Maru J, Kato H, Nakamura A, Shinohara N, Uchino K, Honda K - Inhal Toxicol (2015)

Characterization of SWCNTs dispersed in working solutions. Characterization of SWCNTs in working solutions for in vivo and in vitro tests (A). TEM images of SWCNTs in CNT-1 (B and C) and CNT-2 (D and E) in working solutions for in vivo (high dose) studies. High-magnification micrographs (C and E). Distribution of SWCNT length in working solutions for in vivo (high dose) evaluation by digital TEM images (F and G).
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Figure 0001: Characterization of SWCNTs dispersed in working solutions. Characterization of SWCNTs in working solutions for in vivo and in vitro tests (A). TEM images of SWCNTs in CNT-1 (B and C) and CNT-2 (D and E) in working solutions for in vivo (high dose) studies. High-magnification micrographs (C and E). Distribution of SWCNT length in working solutions for in vivo (high dose) evaluation by digital TEM images (F and G).
Mentions: We prepared two types of dispersions of SWCNTs with different sizes to elucidate the effect of size on in vivo and in vitro toxicities. The characterization of SWCNTs in working solutions for in vivo and in vitro tests is shown in Figure 1. Analysis with an absorption spectrometer demonstrated nearly equivalent SWCNT concentrations in the adjusted working solutions (approximately 0.1 mg/mL and 1.0 mg/mL for in vivo tests; approximately 0.1 mg/mL for in vitro cell-based assays). DLS and zeta-potential studies revealed the average diameters of the dispersed SWCNT particles and the dispersibility/aggregability characteristics of CNT-1 and CNT-2. These analyses confirmed that SWCNT particles were stable and dispersed in the working solutions. DLS results confirmed that no significant changes occurred in the light scattering intensity-averaged diameter of SWCNTs in cell culture medium for 3 d after preparation (data not shown); thus, working solutions were used within 3 d after preparation for in vitro cell-based assays. Furthermore, no significant difference was found in the average diameter of SWCNTs in both stock suspensions stored at 4 °C for 8 weeks after preparation (data not shown). Working solutions were used within 7 d after preparation for in vivo tests. The negative zeta potential of all adjusted samples revealed that SWCNT particles were well dispersed in working solutions. Raman spectrophotometric analysis revealed that the G/D ratio for bulk SWCNTs was reduced slightly, from 5.9 to 4.1 or 2.4 for CNT-1 or CNT-2, respectively. ICP-MS results revealed that the metal content (e.g. Fe, Y, and Ni) of CNT-1 and CNT-2 was at an undetectable level. Previously, we demonstrated that the adsorption of proteins by ultrafine metal oxide particles influences their cytotoxicity to cultured cells (Horie et al., 2009). There was no significant reduction of salt concentrations (e.g. Na, P, and Ca) in CNT-1 and CNT-2 in culture medium (data not shown). TEM images of representative SWCNT dispersions showed differences in their lengths in working solutions, designated here as CNT-1 and CNT-2. CNT-1 was observed to contain relatively thick SWCNT structures with a short linear shape (Figure 1A, B, and F). The nanotubes in the CNT-2 working solution assembled into relatively thick bundles of SWCNTs with a long linear shape (Figure 1D, E, and G). Noticeably aggregated forms of SWCNTs in each working solution were not identified. These studies revealed that CNT-1 and CNT-2 were stably dispersed and assembled into relatively thick bundles with a long linear shape (CNT-1) or into relatively thin bundles with a short linear shape (CNT-2) in working solutions. The preparation procedure described in this study may thus be valuable for use in both in vivo and in vivo studies.Figure 1.

Bottom Line: Comprehensive gene expression analysis confirmed that CNT-1-induced genes were strongly associated with inflammatory responses, cell proliferation, and immune system processes at 7 or 30 d post-instillation.Numerous genes were significantly upregulated or downregulated by CNT-2 at 1 d post-instillation.CNT-2 treatment induced cell growth inhibition, reactive oxygen species production, MIP-1α expression, and several genes involved in response to stimulus, whereas CNT-1 treatment did not exert a significant impact in these regards.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Science for Safety and Sustainability (RISS), National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba, Ibaraki , Japan .

ABSTRACT
To elucidate the effect of size on the pulmonary toxicity of single-wall carbon nanotubes (SWCNTs), we prepared two types of dispersed SWCNTs, namely relatively thin bundles with short linear shapes (CNT-1) and thick bundles with long linear shapes (CNT-2), and conducted rat intratracheal instillation tests and in vitro cell-based assays using NR8383 rat alveolar macrophages. Total protein levels, MIP-1α expression, cell counts in BALF, and histopathological examinations revealed that CNT-1 caused pulmonary inflammation and slower recovery and that CNT-2 elicited acute lung inflammation shortly after their instillation. Comprehensive gene expression analysis confirmed that CNT-1-induced genes were strongly associated with inflammatory responses, cell proliferation, and immune system processes at 7 or 30 d post-instillation. Numerous genes were significantly upregulated or downregulated by CNT-2 at 1 d post-instillation. In vitro assays demonstrated that CNT-1 and CNT-2 SWCNTs were phagocytized by NR8383 cells. CNT-2 treatment induced cell growth inhibition, reactive oxygen species production, MIP-1α expression, and several genes involved in response to stimulus, whereas CNT-1 treatment did not exert a significant impact in these regards. These results suggest that SWCNTs formed as relatively thin bundles with short linear shapes elicited delayed pulmonary inflammation with slower recovery. In contrast, SWCNTs with a relatively thick bundle and long linear shapes sensitively induced cellular responses in alveolar macrophages and elicited acute lung inflammation shortly after inhalation. We conclude that the pulmonary toxicity of SWCNTs is closely associated with the size of the bundles. These physical parameters are useful for risk assessment and management of SWCNTs.

No MeSH data available.


Related in: MedlinePlus