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Predictive value of in vitro assays depends on the mechanism of toxicity of metal oxide nanoparticles.

Cho WS, Duffin R, Bradley M, Megson IL, MacNee W, Lee JK, Jeong J, Donaldson K - Part Fibre Toxicol (2013)

Bottom Line: In this study, we used 9 different NPs (CeO2, TiO2, carbon black, SiO2, NiO, Co3O4, Cr2O3, CuO, and ZnO).As an in vivo toxicity endpoint, the acute lung inflammogenicity in a rat instillation model was compared with the in vitro toxicity endpoints comprising cytotoxicity, pro-inflammatory cytokine expression, or haemolytic potential.Cytotoxicity in differentiated peripheral blood mononuclear cells was the most accurate showing 89% accuracy and 11% false negativity in predicting acute lung inflammogenicity.

View Article: PubMed Central - HTML - PubMed

Affiliation: ELEGI/Colt Laboratory, Centre for Inflammation Research, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK. ken.donaldson@ed.ac.uk.

ABSTRACT

Background: Hazard identification for risk assessment of nanoparticles (NPs) is mainly composed of in vitro cell-based assays and in vivo animal experimentation. The rapidly increasing number and functionalizations of NPs makes in vivo toxicity tests undesirable on both ethical and financial grounds, creating an urgent need for development of in vitro cell-based assays that accurately predict in vivo toxicity and facilitate safe nanotechnology.

Methods: In this study, we used 9 different NPs (CeO2, TiO2, carbon black, SiO2, NiO, Co3O4, Cr2O3, CuO, and ZnO). As an in vivo toxicity endpoint, the acute lung inflammogenicity in a rat instillation model was compared with the in vitro toxicity endpoints comprising cytotoxicity, pro-inflammatory cytokine expression, or haemolytic potential. For in vitro assays, 8 different cell-based assays were used including epithelial cells, monocytic/macrophage cells, human erythrocytes, and combined culture.

Results: ZnO and CuO NPs acting via soluble toxic ions showed positive results in most of assays and were consistent with the lung inflammation data. When compared in in vitro assays at the same surface area dose (30 cm2/mL), NPs that were low solubility and therefore acting via surface reactivity had no convincing activity, except for CeO2 NP. Cytotoxicity in differentiated peripheral blood mononuclear cells was the most accurate showing 89% accuracy and 11% false negativity in predicting acute lung inflammogenicity. However, the haemolysis assay showed 100% consistency with the lung inflammation if any dose, having statistical significance was considered positivity. Other cell-based in vitro assays showed a poorer correlation with in vivo inflammogenicity.

Conclusions: Based on the toxicity mechanisms of NPs, two different approaches can be applied for prediction of in vivo lung inflammogenicity. Most in vitro assays were good at detecting NPs that act via soluble ions (i.e., ZnO and CuO NP). However, in vitro assays were limited in detecting NPs acting via surface reactivity as their mechanism of toxicity, except for the haemolysis assay.

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Cytotoxicity and IL-8 expression of A549 cells after exposure to NPs for 24 h. (A) Cytotoxicity was measured by trypan blue exclusion for ZnO and CuO NP whilst others were measured by LDH. (B) Levels of IL-8 of A549 cells at 24 h following treatment. Note that the surface area doses were 30, 100, and 300 cm2/mL except for ZnO and CuO NP which were 3, 10, and 30 cm2/mL. Values are mean ± SD from minimum four independent experiments. Significance versus vehicle control (VEH): *p < 0.05, ***p < 0.001.
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Figure 1: Cytotoxicity and IL-8 expression of A549 cells after exposure to NPs for 24 h. (A) Cytotoxicity was measured by trypan blue exclusion for ZnO and CuO NP whilst others were measured by LDH. (B) Levels of IL-8 of A549 cells at 24 h following treatment. Note that the surface area doses were 30, 100, and 300 cm2/mL except for ZnO and CuO NP which were 3, 10, and 30 cm2/mL. Values are mean ± SD from minimum four independent experiments. Significance versus vehicle control (VEH): *p < 0.05, ***p < 0.001.

Mentions: Cr2O3, ZnO, and CuO NPs showed significant cytotoxicity (cell death measured by LDH levels or trypan blue staining) compared with vehicle control whilst other NPs had little or no toxic effect in this assay at the doses tested (30–300 cm2/mL for most NPs, 3–30 cm2/mL for ZnO and CuO; Figure 1A). The levels of IL-8 proteins in the supernatant were significantly increased by both ZnO and CuO NP (Figure 1B). Note that ZnO and CuO NP were the most cytotoxic and stimulatory for IL-8 although they were used at doses 10-fold less than the other NPs.


Predictive value of in vitro assays depends on the mechanism of toxicity of metal oxide nanoparticles.

Cho WS, Duffin R, Bradley M, Megson IL, MacNee W, Lee JK, Jeong J, Donaldson K - Part Fibre Toxicol (2013)

Cytotoxicity and IL-8 expression of A549 cells after exposure to NPs for 24 h. (A) Cytotoxicity was measured by trypan blue exclusion for ZnO and CuO NP whilst others were measured by LDH. (B) Levels of IL-8 of A549 cells at 24 h following treatment. Note that the surface area doses were 30, 100, and 300 cm2/mL except for ZnO and CuO NP which were 3, 10, and 30 cm2/mL. Values are mean ± SD from minimum four independent experiments. Significance versus vehicle control (VEH): *p < 0.05, ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016420&req=5

Figure 1: Cytotoxicity and IL-8 expression of A549 cells after exposure to NPs for 24 h. (A) Cytotoxicity was measured by trypan blue exclusion for ZnO and CuO NP whilst others were measured by LDH. (B) Levels of IL-8 of A549 cells at 24 h following treatment. Note that the surface area doses were 30, 100, and 300 cm2/mL except for ZnO and CuO NP which were 3, 10, and 30 cm2/mL. Values are mean ± SD from minimum four independent experiments. Significance versus vehicle control (VEH): *p < 0.05, ***p < 0.001.
Mentions: Cr2O3, ZnO, and CuO NPs showed significant cytotoxicity (cell death measured by LDH levels or trypan blue staining) compared with vehicle control whilst other NPs had little or no toxic effect in this assay at the doses tested (30–300 cm2/mL for most NPs, 3–30 cm2/mL for ZnO and CuO; Figure 1A). The levels of IL-8 proteins in the supernatant were significantly increased by both ZnO and CuO NP (Figure 1B). Note that ZnO and CuO NP were the most cytotoxic and stimulatory for IL-8 although they were used at doses 10-fold less than the other NPs.

Bottom Line: In this study, we used 9 different NPs (CeO2, TiO2, carbon black, SiO2, NiO, Co3O4, Cr2O3, CuO, and ZnO).As an in vivo toxicity endpoint, the acute lung inflammogenicity in a rat instillation model was compared with the in vitro toxicity endpoints comprising cytotoxicity, pro-inflammatory cytokine expression, or haemolytic potential.Cytotoxicity in differentiated peripheral blood mononuclear cells was the most accurate showing 89% accuracy and 11% false negativity in predicting acute lung inflammogenicity.

View Article: PubMed Central - HTML - PubMed

Affiliation: ELEGI/Colt Laboratory, Centre for Inflammation Research, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK. ken.donaldson@ed.ac.uk.

ABSTRACT

Background: Hazard identification for risk assessment of nanoparticles (NPs) is mainly composed of in vitro cell-based assays and in vivo animal experimentation. The rapidly increasing number and functionalizations of NPs makes in vivo toxicity tests undesirable on both ethical and financial grounds, creating an urgent need for development of in vitro cell-based assays that accurately predict in vivo toxicity and facilitate safe nanotechnology.

Methods: In this study, we used 9 different NPs (CeO2, TiO2, carbon black, SiO2, NiO, Co3O4, Cr2O3, CuO, and ZnO). As an in vivo toxicity endpoint, the acute lung inflammogenicity in a rat instillation model was compared with the in vitro toxicity endpoints comprising cytotoxicity, pro-inflammatory cytokine expression, or haemolytic potential. For in vitro assays, 8 different cell-based assays were used including epithelial cells, monocytic/macrophage cells, human erythrocytes, and combined culture.

Results: ZnO and CuO NPs acting via soluble toxic ions showed positive results in most of assays and were consistent with the lung inflammation data. When compared in in vitro assays at the same surface area dose (30 cm2/mL), NPs that were low solubility and therefore acting via surface reactivity had no convincing activity, except for CeO2 NP. Cytotoxicity in differentiated peripheral blood mononuclear cells was the most accurate showing 89% accuracy and 11% false negativity in predicting acute lung inflammogenicity. However, the haemolysis assay showed 100% consistency with the lung inflammation if any dose, having statistical significance was considered positivity. Other cell-based in vitro assays showed a poorer correlation with in vivo inflammogenicity.

Conclusions: Based on the toxicity mechanisms of NPs, two different approaches can be applied for prediction of in vivo lung inflammogenicity. Most in vitro assays were good at detecting NPs that act via soluble ions (i.e., ZnO and CuO NP). However, in vitro assays were limited in detecting NPs acting via surface reactivity as their mechanism of toxicity, except for the haemolysis assay.

Show MeSH
Related in: MedlinePlus