Nano Safety Research Group, School of Engineering and Physical Sciences, Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot-Watt University, Edinburgh, EH14 4AS, UK.
CNR-ISTEC Faenza, Via Granarolo, 64 -, 48018, Faenza, RA, Italy.
Part Fibre Toxicol. 2017 Aug 23;14(1):31. doi: 10.1186/s12989-017-0211-7.
Copper oxide nanomaterials (CuO NMs) are exploited in a diverse array of products including antimicrobials, inks, cosmetics, textiles and food contact materials. There is therefore a need to assess the toxicity of CuO NMs to the gastrointestinal (GI) tract since exposure could occur via direct oral ingestion, mucocillary clearance (following inhalation) or hand to mouth contact.
Undifferentiated Caco-2 intestinal cells were exposed to CuO NMs (10 nm) at concentrations ranging from 0.37 to 78.13 μg/cm Cu (equivalent to 1.95 to 250 μg/ml) and cell viability assessed 24 h post exposure using the alamar blue assay. The benchmark dose (BMD 20), determined using PROAST software, was identified as 4.44 μg/cm for CuO NMs, and 4.25 μg/cm for copper sulphate (CuSO), which informed the selection of concentrations for further studies. The differentiation status of cells and the impact of CuO NMs and CuSO on the integrity of the differentiated Caco-2 cell monolayer were assessed by measurement of trans-epithelial electrical resistance (TEER), staining for Zonula occludens-1 (ZO-1) and imaging of cell morphology using scanning electron microscopy (SEM). The impact of CuO NMs and CuSO on the viability of differentiated cells was performed via assessment of cell number (DAPI staining), and visualisation of cell morphology (light microscopy). Interleukin-8 (IL-8) production by undifferentiated and differentiated Caco-2 cells following exposure to CuO NMs and CuSO was determined using an ELISA. The copper concentration in the cell lysate, apical and basolateral compartments were measured with Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES) and used to calculate the apparent permeability coefficient (P); a measure of barrier permeability to CuO NMs. For all experiments, CuSO was used as an ionic control.
CuO NMs and CuSO caused a concentration dependent decrease in cell viability in undifferentiated cells. CuO NMs and CuSO translocated across the differentiated Caco-2 cell monolayer. CuO NM mediated IL-8 production was over 2-fold higher in undifferentiated cells. A reduction in cell viability in differentiated cells was not responsible for the lower level of cytokine production observed. Both CuO NMs and CuSO decreased TEER values to a similar extent, and caused tight junction dysfunction (ZO-1 staining), suggesting that barrier integrity was disrupted.
CuO NMs and CuSO stimulated IL-8 production by Caco-2 cells, decreased barrier integrity and thereby increased the P and translocation of Cu. There was no significant enhancement in potency of the CuO NMs compared to CuSO. Differentiated Caco-2 cells were identified as a powerful model to assess the impacts of ingested NMs on the GI tract.
氧化铜纳米材料（CuO NMs）在各种产品中得到广泛应用，包括抗菌剂、油墨、化妆品、纺织品和食品接触材料。因此，需要评估 CuO NMs 对胃肠道（GI）的毒性，因为暴露可能通过直接口服、黏液清除（吸入后）或手到口接触而发生。
未分化的 Caco-2 肠细胞在浓度范围为 0.37 至 78.13μg/cm Cu（相当于 1.95 至 250μg/ml）的 CuO NMs 下暴露 24 小时，并使用 alamar blue 测定法评估细胞活力。使用 PROAST 软件确定的基准剂量（BMD 20）为 4.44μg/cm 的 CuO NMs 和 4.25μg/cm 的硫酸铜（CuSO），这为进一步研究选择了浓度。通过测量跨上皮电阻（TEER）、检测封闭蛋白-1（ZO-1）的染色和扫描电子显微镜（SEM）观察细胞形态，评估 CuO NMs 和 CuSO 对分化的 Caco-2 细胞单层完整性的影响。通过评估细胞数量（DAPI 染色）和观察细胞形态（相差显微镜）来评估 CuO NMs 和 CuSO 对分化细胞活力的影响。通过 ELISA 测定暴露于 CuO NMs 和 CuSO 后未分化和分化的 Caco-2 细胞的白细胞介素-8（IL-8）产生。使用电感耦合等离子体发射光谱法（ICP-OES）测量细胞裂解液、顶侧和基底外侧腔室中的铜浓度，并用于计算表观渗透系数（P）；这是衡量 CuO NMs 对屏障通透性的指标。对于所有实验，均使用硫酸铜作为离子对照。
CuO NMs 和 CuSO 导致未分化细胞的细胞活力呈浓度依赖性下降。CuO NMs 和 CuSO 穿过分化的 Caco-2 细胞单层。CuO NM 介导的 IL-8 产生在未分化细胞中增加了两倍以上。分化细胞中细胞活力的降低并不是观察到的细胞因子产生水平降低的原因。CuO NMs 和 CuSO 均降低了 TEER 值，且导致紧密连接功能障碍（ZO-1 染色），表明屏障完整性受到破坏。
CuO NMs 和 CuSO 刺激 Caco-2 细胞产生白细胞介素-8，降低了屏障完整性，从而增加了铜的 P 和转运。与 CuSO 相比，CuO NMs 的效力没有显著增强。分化的 Caco-2 细胞被鉴定为评估摄入的纳米材料对胃肠道影响的有力模型。
