Copper oxide nanomaterials (CuO NMs) are exploited in many products including inks, cosmetics, textiles, wood preservatives and food contact materials. Their incorporation into these products may enhance oral exposure in consumer, environmental and occupational settings. Undifferentiated and differentiated monocultures of Caco-2 cells are commonly used to assess NM toxicity to the intestine in vitro. However, the integration of other cell types into Caco-2 in vitro models increases their physiological relevance. Therefore, the aim of this study is to evaluate the toxicity of CuO NMs and copper sulphate (CuSO4) to intestinal microfold (M) cell (Caco-2/Raji B) and mucus secreting (Caco-2/HT29-MTX) co-culture in vitro models via assessment of their impact on barrier integrity, viability and interleukin (IL)-8 secretion. The translocation of CuO NMs and CuSO4 across the intestinal barrier was also investigated in vitro. CuO NMs and CuSO4 impaired the function of the intestinal barrier in the co-culture models [as indicated by a reduction in transepithelial electrical resistance (TEER) and Zonular occludens (ZO-1) staining intensity]. Cu translocation was observed in both models but was greatest in the Caco-2/Raji B co-culture. CuO NMs and CuSO4 stimulated an increase in IL-8 secretion, which was greatest in the Caco-2/HT29-MTX co-culture model. CuO NMs and CuSO4 did not stimulate a loss of cell viability, when assessed using light microscopy, nuclei counts and scanning electron microscopy. CuO NMs demonstrated a relatively similar level of toxicity to CuO4 in both Caco-2/Raji B and Caco-2/HT29-MTX co- culture models. The Caco-2/Raji B co-culture model was more sensitive to CuO NM and CuSO4 toxicity than the Caco-2/HT29-MTX co-culture model. However, both co-culture models were less sensitive to CuO NM and CuSO4 toxicity than simple monocultures of undifferentiated and differentiated Caco-2 cells, which are more routinely used to investigate NM toxicity to the intestine. Obtained data can therefore feed into the design of future studies which assess the toxicity of substances (e.g. NMs) and pathogens to the intestine (e.g. by informing model and endpoint selection). However, more testing with a wider panel of NMs would be beneficial in order to help select which in vitro models and endpoints to prioritise when screening the safety of ingested NMs. Comparisons with in vivo findings will also be essential to identify the most suitable in vitro model to screen the safety of ingested NMs.

中文翻译：

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利用具有微褶皱细胞和粘液分泌能力的3D胃肠道体外模型评估氧化铜纳米材料的危害

氧化铜纳米材料 (CuO NM) 可用于许多产品，包括油墨、化妆品、纺织品、木材防腐剂和食品接触材料。将它们纳入这些产品中可能会增加消费者、环境和职业环境中的口腔暴露。Caco-2 细胞的未分化和分化单一培养物通常用于评估 NM 对肠道的体外毒性。然而，将其他细胞类型整合到 Caco-2 体外模型中增加了它们的生理相关性。因此，本研究的目的是评估 CuO NMs 和硫酸铜 (CuSO4) 对肠道微褶皱 (M) 细胞 (Caco-2/Raji B) 和粘液分泌细胞 (Caco-2/HT29-MTX) 的毒性。通过评估其对屏障完整性、活力和白细胞介素 (IL)-8 分泌的影响来培养体外模型。CuO NMs 和 CuSO4 穿过肠屏障的易位也在体外进行了研究。CuO NM 和 CuSO4 损害共培养模型中肠屏障的功能 [如跨上皮电阻 (TEER) 和小带闭塞 (ZO-1) 染色强度降低所示]。在两种模型中均观察到铜易位，但在 Caco-2/Raji B 共培养中最为严重。CuO NM 和 CuSO4 刺激 IL-8 分泌增加，这在 Caco-2/HT29-MTX 共培养模型中最为明显。当使用光学显微镜、细胞核计数和扫描电子显微镜进行评估时，CuO NM 和 CuSO4 不会导致细胞活力丧失。CuO NM 在 Caco-2/Raji B 和 Caco-2/HT29-MTX 共培养模型中表现出与 CuO4 相对相似的毒性水平。Caco-2/Raji B 共培养模型比 Caco-2/HT29-MTX 共培养模型对 CuO NM 和 CuSO4 毒性更敏感。然而，与未分化和分化 Caco-2 细胞的简单单一培养相比，两种共培养模型对 CuO NM 和 CuSO4 毒性的敏感性较低，后者更常用于研究 NM 对肠道的毒性。因此，获得的数据可以用于未来研究的设计，评估物质（例如NM）和病原体对肠道的毒性（例如通过告知模型和终点选择）。然而，对更广泛的 NM 进行更多测试将是有益的，以便在筛选摄入 NM 的安全性时帮助选择优先考虑的体外模型和终点。与体内研究结果的比较对于确定最合适的体外模型来筛选摄入的 NM 的安全性也至关重要。