Abstract

The in vitro and in vivo toxicity of copper oxide nanoparticles (CuO NPs) is attributed to both particle and dissolved copper ion species. However, a clear understanding of (1) the specific cellular responses that are modulated by the two species and (2) the temporal dynamics in toxicity, as the proportional amount of particulate and ionic forms change over time, is lacking. In the current study, in vitro responses to microparticulate CuO (CuO MPs), CuO NPs, and dissolved Cu²⁺ were characterized in order to elucidate particle and ion-induced kinetic effects. Particle dissolution experiments were carried out in a relevant cell culture medium, using CuO NPs and MPs. Mouse lung epithelial cells were exposed for 2–48 h with 1–25 µg/mL CuO MPs, CuO NPs, or 7 and 54 µg/mL CuCl₂. Cellular viability and genome-wide transcriptional responses were assessed. Dose and time-dependent cytotoxicity were observed in CuO NP exposed cells, which was delayed and subtle in CuCl₂ and not observed in CuO MPs treated cells. Analyses of differentially expressed genes and associated pathway perturbations showed that dissolved ions released by CuO NPs in the extracellular medium are insufficient to account for the observed potency and cytotoxicity. Further organization of gene expression results in an Adverse Outcome Pathway (AOP) framework revealed a series of key events potentially involved in CuO NPs toxicity. The AOP is applicable to toxicity induced by metal oxide nanoparticles of varying solubility, and thus, can facilitate the development of in vitro alternative strategies to screen their toxicity.

摘要

在体外和体内的氧化铜纳米粒子（CuO的NPS）的毒性是由于两个粒子和溶解的铜离子种。然而，由于颗粒和离子形式的比例随时间变化，因此缺乏对（1）由两种物质调节的特定细胞应答和（2）毒性的时间动态的清晰了解。在当前的研究中，表征了对微粒CuO（CuO MPs），CuO NPs和溶解的Cu ^2+的体外反应，以阐明颗粒和离子诱导的动力学效应。使用CuO NP和MP在相关的细胞培养基中进行了颗粒溶解实验。小鼠肺上皮细胞暴露2-48 h为1–25 µg / mL CuO MP，CuO NP或7和54 µg / mL CuCl ₂。评估了细胞活力和全基因组转录反应。在暴露于CuO NP的细胞中观察到剂量和时间依赖性的细胞毒性，在CuCl _2中延迟并微妙在CuO MPs处理的细胞中未观察到。对差异表达基因和相关途径扰动的分析表明，CuO NPs在细胞外培养基中释放的溶解离子不足以说明所观察到的效力和细胞毒性。基因表达结果在不良结果途径（AOP）框架中的进一步组织揭示了一系列可能与CuO NPs毒性有关的关键事件。AOP适用于溶解度各不相同的金属氧化物纳米粒子引起的毒性，因此，可以促进开发筛选其毒性的体外替代策略。