The terms agglomerates and aggregates are frequently used in the regulatory definition(s) of nanomaterials (NMs) and hence attract attention in view of their potential influence on health effects. However, the influence of nanoparticle (NP) agglomeration and aggregation on toxicity is poorly understood although it is strongly believed that smaller the size of the NPs greater the toxicity. A toxicologically relevant definition of NMs is therefore not yet available, which affects not only the risk assessment process but also hinders the regulation of nano-products. In this study, we assessed the influence of NP agglomeration on their toxicity/biological responses in vitro and in vivo. We tested two TiO2 NPs with different primary sizes (17 and 117 nm) and prepared ad-hoc suspensions composed of small or large agglomerates with similar dispersion medium composition. For in vitro testing, human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic (THP-1) cell lines were exposed to these suspensions for 24 h and endpoints such as cytotoxicity, total glutathione, epithelial barrier integrity, inflammatory mediators and DNA damage were measured. Large agglomerates of 17 nm TiO2 induced stronger responses than small agglomerates for glutathione depletion, IL-8 and IL-1β increase, and DNA damage in THP-1, while no effect of agglomeration was observed with 117 nm TiO2. In vivo, C57BL/6JRj mice were exposed via oropharyngeal aspiration or oral gavage to TiO2 suspensions and, after 3 days, biological parameters including cytotoxicity, inflammatory cell recruitment, DNA damage and biopersistence were measured. Mainly, we observed that large agglomerates of 117 nm TiO2 induced higher pulmonary responses in aspirated mice and blood DNA damage in gavaged mice compared to small agglomerates. Agglomeration of TiO2 NPs influences their toxicity/biological responses and, large agglomerates do not appear less active than small agglomerates. This study provides a deeper insight on the toxicological relevance of NP agglomerates and contributes to the establishment of a toxicologically relevant definition for NMs.

中文翻译：

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二氧化钛纳米颗粒的附聚增加了体外和体内的毒理学反应。

术语团聚物和团聚物经常用于纳米材料（NMs）的法规定义中，因此鉴于其对健康影响的潜在影响而引起关注。然而，尽管人们强烈认为纳米颗粒（NP）团聚和聚集对毒性的影响较小，但人们对其了解甚少。因此，尚无关于NM的毒理学相关定义，这不仅影响风险评估过程，而且阻碍了纳米产品的监管。在这项研究中，我们评估了NP团聚对其在体外和体内的毒性/生物学反应的影响。我们测试了两种具有不同主要尺寸（17和117 nm）的TiO2 NP，并制备了由具有相似分散介质组成的小或大团聚物组成的临时悬浮液。为了进行体外测试，将人支气管上皮（HBE），结肠上皮（Caco2）和单核细胞（THP-1）细胞系暴露于这些悬浮液24小时，并观察终点，例如细胞毒性，总谷胱甘肽，上皮屏障完整性，炎性介质和测量DNA损伤。17 nm TiO2的大团聚体对谷胱甘肽耗竭的反应强于小团聚体，IL-8和IL-1β增加，THP-1中的DNA损伤，而117 nm TiO2则未观察到团聚作用。在体内，通过口咽抽吸或口服管饲法将C57BL / 6JRj小鼠暴露于TiO2悬浮液中，并在3天后，测量了包括细胞毒性，炎症细胞募集，DNA损伤和生物持久性在内的生物学参数。主要是，我们观察到，与小团聚物相比，117 nm TiO2的大团聚物在吸气小鼠中诱导了更高的肺反应，在空腹小鼠中引起了血液DNA损伤。TiO2 NPs的团聚会影响其毒性/生物反应，而且大的团聚体的活性似乎不如小的团聚体低。这项研究为NP团聚体的毒理学相关性提供了更深入的见解，并有助于建立NMs的毒理学相关定义。我们观察到，与小团聚体相比，117 nm TiO2的大团聚体在吸气小鼠中诱导了更高的肺部反应，在空腹小鼠中引起血液DNA损伤。TiO2 NPs的团聚会影响其毒性/生物反应，而且大的团聚体的活性似乎不如小的团聚体低。这项研究为NP团聚体的毒理学相关性提供了更深入的见解，并有助于建立NMs的毒理学相关定义。我们观察到，与小团聚体相比，117 nm TiO2的大团聚体在吸气小鼠中诱导了更高的肺部反应，在空腹小鼠中引起血液DNA损伤。TiO2 NPs的团聚会影响其毒性/生物反应，并且大的团聚体的活性似乎不如小的团聚体低。这项研究为NP团聚体的毒理学相关性提供了更深入的见解，并有助于建立NMs的毒理学相关定义。