Genetic toxicity assessment of engineered nanoparticles using a 3D in vitro skin model (EpiDerm™).
Genetic toxicity assessment of engineered nanoparticles using a 3D in vitro skin model (EpiDerm™).
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DOI：
10.1186/s12989-016-0161-5
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发表时间：
2016-09-09
影响因子：
10
通讯作者：
Doak SH
中科院分区：
文献类型：
--
作者：
Wills JW;Hondow N;Thomas AD;Chapman KE;Fish D;Maffeis TG;Penny MW;Brown RA;Jenkins GJ;Brown AP;White PA;Doak SH
工程纳米材料的快速生产和融入到消费品中，以及研究表明纳米材料可能导致细胞死亡和DNA损伤（遗传毒性），使得纳米安全筛选的体外测定成为必要。然而，当比较体外和体内研究结果时，经常观察到相互矛盾的结果，这表明需要更多具有生理代表性的体外模型来最大限度地减少对动物试验的依赖。使用BASF Levasil®二氧化硅纳米颗粒（16和85 nm）来适应3D重建皮肤微核（RSMN）试验，用于局部施用纳米材料或将纳米材料放入生长培养基中。通过总纳米颗粒质量与细胞数量对2D / 3D测定的剂量进行标准化，将3D剂量反应与使用单培养人B细胞（TK6）的2D微核测定进行比较。低温玻璃化，扫描电子显微镜和动态光散射技术应用于表征介质和空气-液体界面暴露。采用先进的透射电子显微镜成像模式（高角度环形暗场）和x射线光谱法来定义完整3D模型和2D单培养细胞中的纳米颗粒穿透/细胞摄取。对于所有2D暴露，在剂量≥200 μg/mL （16 nm-SiO2）和≥100 μg/mL （85 nm-SiO2）时，观察到遗传毒性显著（p < 0.002）增加（≥100 μg/mL），细胞活力降低（p < 0.015）。相比之下，3D等效暴露于3D模型（≤300 μg/mL）没有造成明显的DNA损伤或对细胞活力的影响。对三维模型进一步增加剂量可能导致气液界面窒息。纳米颗粒渗透/细胞摄取分析显示，由于皮肤模型的3D细胞微结构的保护性质（局部暴露）和胶原细胞附着层的混杂屏障效应（中等暴露），没有发生3D模型的活细胞暴露。2D单培养细胞同时显示两种二氧化硅颗粒引起（基因）毒性的广泛内化。研究结果证实了组织微结构在确定纳米材料暴露方面的重要性，并表明3D体外模型可以在纳米毒理学中弥合体外和体内结果之间的差距。稳健的暴露表征和摄取评估方法（如所示）对于成功解释纳米（基因）毒性研究至关重要。本文的在线版本（doi:10.1186/s12989-016-0161-5）包含补充材料，可供授权用户使用。
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. 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. 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. 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. The online version of this article (doi:10.1186/s12989-016-0161-5) contains supplementary material, which is available to authorized users.
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