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

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® 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.


Related in: MedlinePlus

Characterising 3D topical nanosilica exposures after deposition in acetone using cryogenic vitrification and scanning electron microscopy: Representative images 16 nm-SiO2 (a – d), 85 nm-SiO2 (f – i). Particle deposition (false coloured red) varied and was heterogeneous across the tissue surface. Surface coverage (median = bars, error = range) is summarised in e/j (n = 5). Increasing dose (rows) typically resulted in greater / deeper surface coverage, with only the far peripheries of the tissue remaining unexposed to nanoparticles at 1000 μg (d, i). Alternative dose metrics including the 3D equivalent total mass doses with area unit components are provided in Table 2
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Fig4: Characterising 3D topical nanosilica exposures after deposition in acetone using cryogenic vitrification and scanning electron microscopy: Representative images 16 nm-SiO2 (a – d), 85 nm-SiO2 (f – i). Particle deposition (false coloured red) varied and was heterogeneous across the tissue surface. Surface coverage (median = bars, error = range) is summarised in e/j (n = 5). Increasing dose (rows) typically resulted in greater / deeper surface coverage, with only the far peripheries of the tissue remaining unexposed to nanoparticles at 1000 μg (d, i). Alternative dose metrics including the 3D equivalent total mass doses with area unit components are provided in Table 2

Mentions: To characterise 3D topical exposures, particle deposition state was preserved immediately after dosing by vitrification in liquid nitrogen [43], and was subsequently imaged using cryogenic scanning electron microscopy (cryo-SEM) (Fig. 4). For both the 16 nm-SiO2 and 85 nm-SiO2, the lower total mass doses (≤300 μg) resulted in patchy, heterogenous surface coverage, with some areas of the tissue surface found to remain completely free of nanoparticles. With increasing dose (≥450 μg) however, coverage became increasingly layered and thick enough to mask the surface features of the underlying tissue. Areas of unexposed stratum corneum became less common and were found only at the far peripheries of the tissue surface for the highest (1000 μg) exposures. Further cryo-SEM images are presented in Additional files 5 and 6.Fig. 4


Genetic toxicity assessment of engineered nanoparticles using a 3D in vitro skin model (EpiDerm ™ )
Characterising 3D topical nanosilica exposures after deposition in acetone using cryogenic vitrification and scanning electron microscopy: Representative images 16 nm-SiO2 (a – d), 85 nm-SiO2 (f – i). Particle deposition (false coloured red) varied and was heterogeneous across the tissue surface. Surface coverage (median = bars, error = range) is summarised in e/j (n = 5). Increasing dose (rows) typically resulted in greater / deeper surface coverage, with only the far peripheries of the tissue remaining unexposed to nanoparticles at 1000 μg (d, i). Alternative dose metrics including the 3D equivalent total mass doses with area unit components are provided in Table 2
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5016964&req=5

Fig4: Characterising 3D topical nanosilica exposures after deposition in acetone using cryogenic vitrification and scanning electron microscopy: Representative images 16 nm-SiO2 (a – d), 85 nm-SiO2 (f – i). Particle deposition (false coloured red) varied and was heterogeneous across the tissue surface. Surface coverage (median = bars, error = range) is summarised in e/j (n = 5). Increasing dose (rows) typically resulted in greater / deeper surface coverage, with only the far peripheries of the tissue remaining unexposed to nanoparticles at 1000 μg (d, i). Alternative dose metrics including the 3D equivalent total mass doses with area unit components are provided in Table 2
Mentions: To characterise 3D topical exposures, particle deposition state was preserved immediately after dosing by vitrification in liquid nitrogen [43], and was subsequently imaged using cryogenic scanning electron microscopy (cryo-SEM) (Fig. 4). For both the 16 nm-SiO2 and 85 nm-SiO2, the lower total mass doses (≤300 μg) resulted in patchy, heterogenous surface coverage, with some areas of the tissue surface found to remain completely free of nanoparticles. With increasing dose (≥450 μg) however, coverage became increasingly layered and thick enough to mask the surface features of the underlying tissue. Areas of unexposed stratum corneum became less common and were found only at the far peripheries of the tissue surface for the highest (1000 μg) exposures. Further cryo-SEM images are presented in Additional files 5 and 6.Fig. 4

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® 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.


Related in: MedlinePlus