National Research Centre for the Working Environment, DK-2100 Copenhagen, Copenhagen, Denmark.
National Institute of Occupational Health, Oslo, Norway.
Part Fibre Toxicol. 2021 Jul 23;18(1):25. doi: 10.1186/s12989-021-00413-2.
Multi-walled carbon nanotubes (MWCNT) have received attention due to extraordinary properties, resulting in concerns for occupational health and safety. Costs and ethical concerns of animal testing drive a need for in vitro models with predictive power in respiratory toxicity. The aim of this study was to assess pro-inflammatory response (Interleukin-8 expression, IL-8) and genotoxicity (DNA strand breaks) caused by MWCNT with different physicochemical properties in different pulmonary cell models and correlate these to previously published in vivo data. Seven MWCNT were selected; two long/thick (NRCWE-006/Mitsui-7 and NM-401), two short/thin (NM-400 and NM-403), a pristine (NRCWE-040) and two surface modified; hydroxylated (NRCWE-041) and carboxylated (NRCWE-042). Carbon black Printex90 (CB) was included as benchmark material. Human alveolar epithelial cells (A549) and monocyte-derived macrophages (THP-1a) were exposed to nanomaterials (NM) in submerged conditions, and two materials (NM-400 and NM-401) in co-cultures of A549/THP-1a and lung fibroblasts (WI-38) in an air-liquid interface (ALI) system. Effective doses were quantified by thermo-gravimetric-mass spectrometry analysis (TGA-MS). To compare genotoxicity in vitro and in vivo, we developed a scoring system based on a categorization of effects into standard deviation (SD) units (< 1, 1, 2, 3 or 4 standard deviation increases) for the increasing genotoxicity.
Effective doses were shown to be 25 to 53%, and 21 to 57% of the doses administered to A549 and THP-1a, respectively. In submerged conditions (A549 and THP-1a cells), all NM induced dose-dependent IL-8 expression. NM-401 and NRCWE-006 caused the strongest pro-inflammatory response. In the ALI-exposed co-culture, only NM-401 caused increased IL-8 expression, and no DNA strand breaks were observed. Strong correlations were found between in vitro and in vivo inflammation when doses were normalized by surface area (also proxy for diameter and length). Significantly increased DNA damage was found for all MWCNT in THP-1a cells, and for short MWCNT in A549 cells. A concordance in genotoxicity of 83% was obtained between THP-1a cells and broncho-alveolar lavaged (BAL) cells.
This study shows correlations of pro-inflammatory potential in A549 and THP-1a cells with neutrophil influx in mice, and concordance in genotoxic response between THP-1a cells and BAL cells, for seven MWCNT.
多壁碳纳米管（MWCNT）具有非凡的性质，因此引起了人们对职业健康和安全的关注。动物试验的成本和伦理问题促使人们需要具有呼吸毒性预测能力的体外模型。本研究的目的是评估具有不同理化性质的 MWCNT 在不同的肺细胞模型中引起的促炎反应（白细胞介素-8 表达，IL-8）和遗传毒性（DNA 链断裂），并将这些与先前发表的体内数据相关联。选择了七种 MWCNT；两种长而粗（NRCWE-006/Mitsui-7 和 NM-401），两种短而细（NM-400 和 NM-403），一种原始（NRCWE-040）和两种表面改性；羟基化（NRCWE-041）和羧基化（NRCWE-042）。还包括炭黑 Printex90（CB）作为基准材料。将纳米材料（NM）以淹没条件暴露于肺泡上皮细胞（A549）和单核细胞衍生的巨噬细胞（THP-1a）中，并以淹没条件暴露于 NM-400 和 NM-401，在肺泡上皮细胞（A549）/THP-1a 和肺成纤维细胞（WI-38）的共培养物中暴露于空气中-液体界面（ALI）系统。通过热重质量光谱分析（TGA-MS）定量有效剂量。为了比较体外和体内的遗传毒性，我们开发了一种评分系统，该系统基于对效应的分类，分为标准偏差（SD）单位（<1、1、2、3 或 4 个标准差增加），以增加遗传毒性。
显示有效剂量分别为 A549 和 THP-1a 给药剂量的 25%至 53%和 21%至 57%。在淹没条件下（A549 和 THP-1a 细胞），所有 NM 均诱导剂量依赖性的 IL-8 表达。NM-401 和 NRCWE-006 引起最强的促炎反应。在暴露于 ALI 的共培养物中，仅 NM-401 引起 IL-8 表达增加，并且未观察到 DNA 链断裂。当通过表面积归一化（也代表直径和长度）时，发现体外和体内炎症之间存在强烈相关性。在 THP-1a 细胞中，所有 MWCNT 均发现 DNA 损伤明显增加，在 A549 细胞中，短 MWCNT 也发现 DNA 损伤增加。THP-1a 细胞和支气管肺泡灌洗（BAL）细胞之间的遗传毒性一致性为 83%。
本研究表明，A549 和 THP-1a 细胞中的促炎潜力与小鼠中性粒细胞浸润之间存在相关性，并且 THP-1a 细胞和 BAL 细胞之间的遗传毒性反应具有一致性，适用于七种 MWCNT。
