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Genetic toxicity assessment of engineered nanoparticles using a 3D in vitro skin model (EpiDerm ™ )

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ABSTRACT

Background: The rapid production and incorporation of engineered nanomaterials into consumer products alongside research suggesting nanomaterials can cause cell death and DNA damage (genotoxicity) makes in vitro assays desirable for nanosafety screening. However, conflicting outcomes are often observed when in vitro and in vivo study results are compared, suggesting more physiologically representative in vitro models are required to minimise reliance on animal testing.

Method: BASF Levasil® silica nanoparticles (16 and 85 nm) were used to adapt the 3D reconstructed skin micronucleus (RSMN) assay for nanomaterials administered topically or into the growth medium. 3D dose-responses were compared to a 2D micronucleus assay using monocultured human B cells (TK6) after standardising dose between 2D / 3D assays by total nanoparticle mass to cell number. Cryogenic vitrification, scanning electron microscopy and dynamic light scattering techniques were applied to characterise in-medium and air-liquid interface exposures. Advanced transmission electron microscopy imaging modes (high angle annular dark field) and X-ray spectrometry were used to define nanoparticle penetration / cellular uptake in the intact 3D models and 2D monocultured cells.

Results: For all 2D exposures, significant (p < 0.002) increases in genotoxicity were observed (≥100 μg/mL) alongside cell viability decreases (p < 0.015) at doses ≥200 μg/mL (16 nm-SiO2) and ≥100 μg/mL (85 nm-SiO2). In contrast, 2D-equivalent exposures to the 3D models (≤300 μg/mL) caused no significant DNA damage or impact on cell viability. Further increasing dose to the 3D models led to probable air-liquid interface suffocation. Nanoparticle penetration / cell uptake analysis revealed no exposure to the live cells of the 3D model occurred due to the protective nature of the skin model’s 3D cellular microarchitecture (topical exposures) and confounding barrier effects of the collagen cell attachment layer (in-medium exposures). 2D monocultured cells meanwhile showed extensive internalisation of both silica particles causing (geno)toxicity.

Conclusions: The results establish the importance of tissue microarchitecture in defining nanomaterial exposure, and suggest 3D in vitro models could play a role in bridging the gap between in vitro and in vivo outcomes in nanotoxicology. Robust exposure characterisation and uptake assessment methods (as demonstrated) are essential to interpret nano(geno)toxicity studies successfully.

Electronic supplementary material: The online version of this article (doi:10.1186/s12989-016-0161-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


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(Geno)toxicity assessment of silica nanoparticles exposed at equivalent doses to the 2D and 3D test systems: (a) 16 nm-SiO2, and (b) 85 nm-SiO2. BARS = micronucleus frequency; LINES/POINTS = cell viability. 2D cell cultures (2D) (n = 6, error bars = SD)/3D tissues (n = 2, error bars = range; except 1000 μg where n = 1) were exposed for 24 h in absence of cyt B via the 3D topical / in-medium or 2D exposure routes. Genotoxicity was assessed until cell viability decreased below 50 %. Equivalent 2D/3D doses were established by total mass dose normalisation according to the total number of cells in each culture model at time of inoculation (see Methods). (*) (**) (***) indicate statistical significance relative to control at p < 0.05, p < 0.01 and p < 0.001 respectively. Alternative dose metrics including the 3D equivalent total mass doses with area (topical exposures) and volume (in-medium exposures) unit components are provided in Table 2
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Fig6: (Geno)toxicity assessment of silica nanoparticles exposed at equivalent doses to the 2D and 3D test systems: (a) 16 nm-SiO2, and (b) 85 nm-SiO2. BARS = micronucleus frequency; LINES/POINTS = cell viability. 2D cell cultures (2D) (n = 6, error bars = SD)/3D tissues (n = 2, error bars = range; except 1000 μg where n = 1) were exposed for 24 h in absence of cyt B via the 3D topical / in-medium or 2D exposure routes. Genotoxicity was assessed until cell viability decreased below 50 %. Equivalent 2D/3D doses were established by total mass dose normalisation according to the total number of cells in each culture model at time of inoculation (see Methods). (*) (**) (***) indicate statistical significance relative to control at p < 0.05, p < 0.01 and p < 0.001 respectively. Alternative dose metrics including the 3D equivalent total mass doses with area (topical exposures) and volume (in-medium exposures) unit components are provided in Table 2

Mentions: 2D and 3D responses to the 16 nm-SiO2 and 85 nm-SiO2 in terms of relative cell viability and binucleated cell MN frequency are presented in Fig. 6. Significant decreases in cell viability were found in the 2D assays at doses ≥200 μg/mL for the 16 nm-SiO2 (p < 0.0016) and ≥100 μg/mL for the 85 nm-SiO2 (p < 0.015). Furthermore, significant MN induction was found for all exposures of both particles (p < 0.002). Equivalent 3D exposures had no significant effect on 3D model viability or MN frequency regardless of exposure route (up to 450 μg) (p > 0.38). For this reason, a single 3D replicate dosed at 1000 μg was also examined. At this extreme, well above the 50 % cytotoxicity threshold for both particle types at equivalent dose in the 2D assay, no (geno)toxic response was observed for the 3D topical 16 nm-SiO2 and 3D in-medium 85 nm-SiO2 exposures. At this dose however, a small decrease in cell viability (88 % of control) and accompanying rise in MN frequency (2.8 fold) was detected for the 16 nm-SiO2 in-medium, and a sharp decline in cell viability (44 %) was noted for the 85 nm-SiO2 topical exposure. Due to the single replicate nature of these results, statistical analysis was not attempted and they are instead presented as preliminary findings to promote discussions regarding the importance of cellular uptake assessment in the avoidance of false positive results in 3D assays. The dose-response data used in the creation of Fig. 6 are provided in Additional file 7.Fig. 6


Genetic toxicity assessment of engineered nanoparticles using a 3D in vitro skin model (EpiDerm ™ )
(Geno)toxicity assessment of silica nanoparticles exposed at equivalent doses to the 2D and 3D test systems: (a) 16 nm-SiO2, and (b) 85 nm-SiO2. BARS = micronucleus frequency; LINES/POINTS = cell viability. 2D cell cultures (2D) (n = 6, error bars = SD)/3D tissues (n = 2, error bars = range; except 1000 μg where n = 1) were exposed for 24 h in absence of cyt B via the 3D topical / in-medium or 2D exposure routes. Genotoxicity was assessed until cell viability decreased below 50 %. Equivalent 2D/3D doses were established by total mass dose normalisation according to the total number of cells in each culture model at time of inoculation (see Methods). (*) (**) (***) indicate statistical significance relative to control at p < 0.05, p < 0.01 and p < 0.001 respectively. Alternative dose metrics including the 3D equivalent total mass doses with area (topical exposures) and volume (in-medium exposures) unit components are provided in Table 2
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Fig6: (Geno)toxicity assessment of silica nanoparticles exposed at equivalent doses to the 2D and 3D test systems: (a) 16 nm-SiO2, and (b) 85 nm-SiO2. BARS = micronucleus frequency; LINES/POINTS = cell viability. 2D cell cultures (2D) (n = 6, error bars = SD)/3D tissues (n = 2, error bars = range; except 1000 μg where n = 1) were exposed for 24 h in absence of cyt B via the 3D topical / in-medium or 2D exposure routes. Genotoxicity was assessed until cell viability decreased below 50 %. Equivalent 2D/3D doses were established by total mass dose normalisation according to the total number of cells in each culture model at time of inoculation (see Methods). (*) (**) (***) indicate statistical significance relative to control at p < 0.05, p < 0.01 and p < 0.001 respectively. Alternative dose metrics including the 3D equivalent total mass doses with area (topical exposures) and volume (in-medium exposures) unit components are provided in Table 2
Mentions: 2D and 3D responses to the 16 nm-SiO2 and 85 nm-SiO2 in terms of relative cell viability and binucleated cell MN frequency are presented in Fig. 6. Significant decreases in cell viability were found in the 2D assays at doses ≥200 μg/mL for the 16 nm-SiO2 (p < 0.0016) and ≥100 μg/mL for the 85 nm-SiO2 (p < 0.015). Furthermore, significant MN induction was found for all exposures of both particles (p < 0.002). Equivalent 3D exposures had no significant effect on 3D model viability or MN frequency regardless of exposure route (up to 450 μg) (p > 0.38). For this reason, a single 3D replicate dosed at 1000 μg was also examined. At this extreme, well above the 50 % cytotoxicity threshold for both particle types at equivalent dose in the 2D assay, no (geno)toxic response was observed for the 3D topical 16 nm-SiO2 and 3D in-medium 85 nm-SiO2 exposures. At this dose however, a small decrease in cell viability (88 % of control) and accompanying rise in MN frequency (2.8 fold) was detected for the 16 nm-SiO2 in-medium, and a sharp decline in cell viability (44 %) was noted for the 85 nm-SiO2 topical exposure. Due to the single replicate nature of these results, statistical analysis was not attempted and they are instead presented as preliminary findings to promote discussions regarding the importance of cellular uptake assessment in the avoidance of false positive results in 3D assays. The dose-response data used in the creation of Fig. 6 are provided in Additional file 7.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: The rapid production and incorporation of engineered nanomaterials into consumer products alongside research suggesting nanomaterials can cause cell death and DNA damage (genotoxicity) makes in vitro assays desirable for nanosafety screening. However, conflicting outcomes are often observed when in vitro and in vivo study results are compared, suggesting more physiologically representative in vitro models are required to minimise reliance on animal testing.

Method: BASF Levasil&reg; silica nanoparticles (16 and 85&nbsp;nm) were used to adapt the 3D reconstructed skin micronucleus (RSMN) assay for nanomaterials administered topically or into the growth medium. 3D dose-responses were compared to a 2D micronucleus assay using monocultured human B cells (TK6) after standardising dose between 2D / 3D assays by total nanoparticle mass to cell number. Cryogenic vitrification, scanning electron microscopy and dynamic light scattering techniques were applied to characterise in-medium and air-liquid interface exposures. Advanced transmission electron microscopy imaging modes (high angle annular dark field) and X-ray spectrometry were used to define nanoparticle penetration / cellular uptake in the intact 3D models and 2D monocultured cells.

Results: For all 2D exposures, significant (p&thinsp;&lt;&thinsp;0.002) increases in genotoxicity were observed (&ge;100&nbsp;&mu;g/mL) alongside cell viability decreases (p&thinsp;&lt;&thinsp;0.015) at doses &ge;200&nbsp;&mu;g/mL (16&nbsp;nm-SiO2) and &ge;100&nbsp;&mu;g/mL (85&nbsp;nm-SiO2). In contrast, 2D-equivalent exposures to the 3D models (&le;300&nbsp;&mu;g/mL) caused no significant DNA damage or impact on cell viability. Further increasing dose to the 3D models led to probable air-liquid interface suffocation. Nanoparticle penetration / cell uptake analysis revealed no exposure to the live cells of the 3D model occurred due to the protective nature of the skin model&rsquo;s 3D cellular microarchitecture (topical exposures) and confounding barrier effects of the collagen cell attachment layer (in-medium exposures). 2D monocultured cells meanwhile showed extensive internalisation of both silica particles causing (geno)toxicity.

Conclusions: The results establish the importance of tissue microarchitecture in defining nanomaterial exposure, and suggest 3D in vitro models could play a role in bridging the gap between in vitro and in vivo outcomes in nanotoxicology. Robust exposure characterisation and uptake assessment methods (as demonstrated) are essential to interpret nano(geno)toxicity studies successfully.

Electronic supplementary material: The online version of this article (doi:10.1186/s12989-016-0161-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus