Farcal Lucian, Torres Andón Fernando, Di Cristo Luisana, Rotoli Bianca Maria, Bussolati Ovidio, Bergamaschi Enrico, Mech Agnieszka, Hartmann Nanna B, Rasmussen Kirsten, Riego-Sintes Juan, Ponti Jessica, Kinsner-Ovaskainen Agnieszka, Rossi François, Oomen Agnes, Bos Peter, Chen Rui, Bai Ru, Chen Chunying, Rocks Louise, Fulton Norma, Ross Bryony, Hutchison Gary, Tran Lang, Mues Sarah, Ossig Rainer, Schnekenburger Jürgen, Campagnolo Luisa, Vecchione Lucia, Pietroiusti Antonio, Fadeel Bengt
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.
PLoS One. 2015 May 21;10(5):e0127174. doi: 10.1371/journal.pone.0127174. eCollection 2015.
Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry - hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO - uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques - precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially 'weak-embryotoxic' and ZnO and SiO2 NMs as 'non-embryotoxic'. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.
纳米材料（NMs）具有许多独特且有用的物理化学性质。然而，需要可靠的方法来对纳米材料进行风险评估。本研究是在FP7-MARINA项目中开展的，目的是识别和评估用于毒性评估的体外测试方法，以促进智能测试策略（ITS）的开发。欧洲委员会联合研究中心纳米材料库提供的六种代表性氧化物纳米材料在九个实验室进行了测试。在代表6种不同靶器官/系统（免疫系统、呼吸系统、胃肠道系统、生殖器官、肾脏和胚胎组织）的12种细胞模型中评估了纳米材料的体外毒性。使用10种不同的细胞毒性、胚胎毒性、上皮完整性、细胞因子分泌和氧化应激检测方法进行毒性评估。对所有测试的纳米材料进行了全面的物理化学表征。选择了具有不同物理化学性质的具有商业相关性的纳米材料：两种具有不同表面化学性质的二氧化钛纳米材料——亲水性（NM-103）和疏水性（NM-104），两种形式的氧化锌——未涂层的（NM-110）和涂有三乙氧基辛基硅烷的（NM-111），以及两种通过两种不同制造技术生产的二氧化硅纳米材料——沉淀法（NM-200）和热解法（NM-203）。观察到了所有纳米材料的细胞特异性毒性作用；短期暴露（24 - 72小时）后，巨噬细胞是最敏感的细胞类型（氧化锌>二氧化硅>二氧化钛）。长期暴露（7至21天）在存在氧化锌的情况下显著影响细胞屏障完整性，但二氧化钛和二氧化硅则不然，而胚胎干细胞试验（EST）将二氧化钛纳米材料归类为潜在的“弱胚胎毒性”，氧化锌和二氧化硅纳米材料归类为“非胚胎毒性”。可以为测试的代表性纳米材料建立危害等级（氧化锌NM-110 > 氧化锌NM-111 > 二氧化硅NM-203 > 二氧化硅NM-200 > 二氧化钛NM-104 > 二氧化钛NM-103）。在胚胎组织的情况下，该等级有所不同，其中二氧化钛显示出比氧化锌和二氧化硅更高的毒性。重要的是，所应用的体外方法可以识别细胞和纳米材料特异性反应，不同测试方法之间观察到低变异性。总体而言，这种基于一系列细胞系统和测试方法，并辅以纳米材料详尽物理化学表征的测试方法，可用于开发适用于纳米材料风险评估的智能测试策略。本研究还为纳米材料效应建模提供了丰富的数据来源。
