Titanium dioxide nanoparticles (TiO2-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1-50 μg/mL TiO2-NPs, 86% anatase/14% rutile, 24 ± 6 nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO2-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO2-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO2-NPs.

中文翻译：

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A549细胞长期暴露于二氧化钛纳米颗粒会引起DNA损伤，并使细胞对遗传毒性剂敏感。

二氧化钛纳米颗粒（TiO2-NPs）是世界上生产量最大的NPs之一。使用急性暴露场景，即高暴露浓度和短暴露时间，已经对其毒性进行了十年的研究。在本研究中，我们使用长期和低浓度暴露条件评估了它们的遗传毒性影响。将A549肺泡上皮细胞连续暴露于1-50μg/ mL TiO2-NP，86％锐钛矿/ 14％金红石，平均一次直径为24±6 nm的情况下，长达两个月。使用MTT分析和活性氧测量，透射电子显微镜观察，微粒诱导的X射线发射和电感耦合等离子体质谱法评估了它们的细胞毒性，氧化电位和细胞内积累。使用碱性和Fpg修饰的彗星试验评估了遗传毒性影响，53BP1病灶的免疫染色和胞质阻滞微核检测。最后，我们评估了这些细胞随后暴露于烷基化剂甲磺酸甲酯的影响。我们证明，长期暴露于TiO2-NPs不会影响细胞活力，但会导致DNA损伤，特别是对DNA的氧化损伤和增加的53BP1病灶数，与增加的NPs的细胞内积累有关。另外，暴露超过2个月会引起细胞适应性反应，其特征在于增殖速率降低和TiO2-NP细胞内积累稳定，以及对MMS致敏。总之，这些数据强调了长期暴露于低水平的TiO2-NPs的肺泡上皮细胞的遗传毒性影响和致敏作用。